Nucleic acid products and methods of administration thereof

ABSTRACT

The present invention relates in part to nucleic acids, including nucleic acids encoding proteins, therapeutics and cosmetics comprising nucleic acids, methods for delivering nucleic acids to cells, tissues, organs, and patients, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, therapeutics, and cosmetics produced using these methods, kits, and devices.

PRIORITY

This application is a continuation of International ApplicationPCT/US2017/047440, filed Aug. 17, 2017. PCT/US2017/047440 claimspriority to U.S. Provisional Patent Application No. 62/376,209, filedAug. 17, 2016 and to U.S. Provisional Patent Application No. 62/509,350,filed May 22, 2017. The entire contents of the aforementioned patentapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, in part, to methods, compositions, andproducts for producing and delivering nucleic acids to cells, tissues,organs, and patients, methods for expressing proteins in cells, tissues,organs, and patients, and cells, therapeutics, and cosmetics producedusing these methods, compositions, and products.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, filed Jan. 26, 2018 is named“FAB-010US-Sequence_Listing_ST25.txt” and is 2,439,641 bytes in size.

BACKGROUND

Synthetic RNA and Nucleic-Acid Therapeutics

Ribonucleic acid (RNA) is ubiquitous in both prokaryotic and eukaryoticcells, where it encodes genetic information in the form of messengerRNA, binds and transports amino acids in the form of transfer RNA,assembles amino acids into proteins in the form of ribosomal RNA, andperforms numerous other functions including gene expression regulationin the forms of microRNA and long non-coding RNA. RNA can be producedsynthetically by methods including direct chemical synthesis and invitro transcription, and can be administered to patients for therapeuticuse. However, previously described synthetic RNA molecules are unstableand trigger a potent innate-immune response in human cells. In addition,methods for efficient non-viral delivery of nucleic acids to patients,organs, tissues, and cells in vivo have not been previously described.The many drawbacks of existing synthetic RNA technologies and methodsfor delivery of nucleic acids make them undesirable for therapeutic orcosmetic use.

Cell Reprogramming and Cell-Based Therapies

Cells can be reprogrammed by exposing them to specific extracellularcues and/or by ectopic expression of specific proteins, microRNAs, etc.While several reprogramming methods have been previously described, mostthat rely on ectopic expression require the introduction of exogenousDNA, which can carry mutation risks. DNA-free reprogramming methodsbased on direct delivery of reprogramming proteins have been reported.However, these methods are too inefficient and unreliable for commercialuse. In addition, RNA-based reprogramming methods have been described(see, e.g., Angel. MIT Thesis. 2008. 1-56; Angel et al. PLoS ONE. 2010.5,107; Warren et al. Cell Stem Cell. 2010. 7, 618-630; Angel. MITThesis. 2011. 1-89; and Lee et al. Cell. 2012. 151, 547-558; thecontents of all of which are hereby incorporated by reference). However,existing RNA-based reprogramming methods are slow, unreliable, andinefficient when performed on adult cells, require many transfections(resulting in significant expense and opportunity for error), canreprogram only a limited number of cell types, can reprogram cells toonly a limited number of cell types, require the use ofimmunosuppressants, and require the use of multiple human-derivedcomponents, including blood-derived HSA and human fibroblast feeders.The many drawbacks of previously disclosed RNA-based reprogrammingmethods make them undesirable for research, therapeutic or cosmetic use.

Gene Editing

Several naturally occurring proteins contain DNA-binding domains thatcan recognize specific DNA sequences, for example, zinc fingers (ZFs)and transcription activator-like effectors (TALEs). Fusion proteinscontaining one or more of these DNA-binding domains and the cleavagedomain of FokI endonuclease can be used to create a double-strand breakin a desired region of DNA in a cell (see, e.g., US Patent Appl. Pub.No. US 2012/0064620, US Patent Appl. Pub. No. US 2011/0239315, U.S. Pat.No. 8,470,973, US Patent Appl. Pub. No. US 2013/0217119, U.S. Pat. No.8,420,782, US Patent Appl. Pub. No. US 2011/0301073, US Patent Appl.Pub. No. US 2011/0145940, U.S. Pat. Nos. 8,450,471, 8,440,431,8,440,432, and US Patent Appl. Pub. No. 2013/0122581, the contents ofall of which are hereby incorporated by reference). Other gene-editingproteins include clustered regularly interspaced short palindromicrepeat (CRISPR)-associated proteins. However, current methods for geneediting cells are inefficient and carry a risk of uncontrolledmutagenesis, making them undesirable for research, therapeutic orcosmetic use. Methods for DNA-free gene editing of somatic cells havenot been previously explored, nor have methods for simultaneous orsequential gene editing and reprogramming of somatic cells. In addition,methods for directly gene editing cells in patients (i.e., in vivo) havenot been previously explored, and the development of such methods hasbeen limited by a lack of acceptable targets, inefficient delivery,inefficient expression of the gene-editing protein/proteins, inefficientgene editing by the expressed gene-editing protein/proteins, due in partto poor binding of DNA-binding domains, excessive off-target effects,due in part to non-directed dimerization of the FokI cleavage domain andpoor specificity of DNA-binding domains, and other factors. Finally, theuse of gene editing in anti-bacterial, anti-viral, and anti-cancertreatments has not been previously explored.

Accordingly, there remains a need for improved methods and compositionsfor the production and delivery of nucleic acids to cells, tissues,organs, and patients.

SUMMARY OF THE INVENTION

The present invention provides, in part, compositions, methods,articles, and devices for delivering nucleic acids to cells, tissues,organs, and patients, methods for inducing cells to express proteins,methods, articles, and devices for producing these compositions,methods, articles, and devices, and compositions and articles, includingcells, organisms, cosmetics and therapeutics, produced using thesecompositions, methods, articles, and devices.

Unlike previously reported methods, certain embodiments of the presentinvention provide small doses of nucleic acids to achieve significantand lasting protein expression in humans.

In some aspects, the present invention relates to a method for treatinga metabolic disorder. The method comprises a step of administering aneffective amount of a synthetic RNA encoding growth differentiationfactor 15 (GDF15) to a subject in need thereof or endothelial cellspecific molecule 1 (ESM1), wherein the synthetic RNA comprises one ormore non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the metabolic disorder is selected from Type Idiabetes, Type II diabetes, insulin resistance, obesity, dyslipidemia,hypercholesterolemia, hyperglycemia, hyperinsulinemia, hypertension,hepatosteaotosis such as non-alcoholic steatohepatitis (NASH) andnon-alcoholic fatty acid liver disease (NAFLD), cancer, a disease ordisorder associated with impaired lipid metabolism, a disease ordisorder associated with impaired renal function, a disease or disorderassociated with impaired hepatic function, a disease or disorderassociated with impaired lung function, a vascular or cardiovasculardisease or disorder, muscle wasting, inflammation, and a respiratorydisease.

In various embodiments, the disease or disorder associated with impairedrenal function is selected from chronic kidney diseases, acute kidneyinjury, nephropathy, diabetic nephropathy, kidney failure, and kidneyfibrosis.

In various embodiments, the vascular or cardiovascular disease ordisorder is selected from coronary artery disease, cardiomyopathy,hypertension, atrial fibrillation, preeclampsia, peripheral arterydisease, atherosclerosis, heart failure, acute myocardial infarction,acute coronary syndrome, muscle wasting, hypertensive ventricularhypertrophy, hypertensive cardiomyopathy, ischemic heart disease,myocardial infarction, abdominal aortic aneurysm, a blood clot, deepvein thrombosis, venous stasis disease, phlebitis, and varicose veins.

In various embodiments, the treatment results in reduction in one ormore of aspartate transaminase (AST), alanine transaminase (ALT), orglucose levels in the subject, as compared to untreated subjects.

In various embodiments, the treatment results in increased lipidmobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,intraportal, rectally, by inhalation, or topically.

In some aspects, the present invention relates to a method formodulating GDF15. The method comprises a step of administering aneffective amount of a synthetic RNA encoding GDF15 to a subject, whereinthe synthetic RNA comprises one or more non-canonical nucleotides thatavoid substantial cellular toxicity.

In various embodiments, the modulating results in reduction in one ormore of ALT, AST, or glucose levels in the subject, as compared tountreated subjects.

In various embodiments, the modulating results in increased lipidmobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,intraportal, rectally, by inhalation, or topically.

In various embodiments, the modulating results in an increase in thequantity of GDF15 in the subject.

In various embodiments, the modulating results in a decrease in thequantity of GDF15 in the subject.

In some aspects, the present invention relates to a method formodulating ESM1. The method comprises a step of administering aneffective amount of a synthetic RNA encoding ESM1 to a subject, whereinthe synthetic RNA comprises one or more non-canonical nucleotides thatavoid substantial cellular toxicity.

In various embodiments, the modulating results in one or more of reducedALT, AST, or glucose levels in the subject, as compared to untreatedsubjects.

In various embodiments, the modulating results in increased lipidmobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,intraportal, rectally, by inhalation, or topically.

In various embodiments, the modulating results in an increase in thequantity of ESM1 in the subject.

In various embodiments, the modulating results in a decrease in thequantity of ESM1 in the subject.

In some aspects, the present invention relates to a method for treatinga liver disorder. The method comprises a step of administering aneffective amount of a synthetic RNA encoding growth differentiationfactor 15 (GDF15) or endothelial cell specific molecule 1 (ESM1) to asubject in need thereof, wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity; andthe treatment reduces one or more of fatty liver, NAFLD, NASH,inflammation, hepatitis, fibrosis, cirrhosis, and hepatocellularcarcinoma in the subject.

In various embodiments, the treatment results in reduction in one ormore of AST, ALT, or glucose levels in the subject, as compared tountreated subjects.

In various embodiments, the treatment results in increased lipidmobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In some aspects, the present invention relates to a method for treatingFriedrich's Ataxia. The method comprises a step of administering to asubject in need thereof an effective amount of (a) a synthetic RNAencoding a gene-editing protein capable of creating a double strandbreak in FXNA (SEQ ID NO: 582), and/or (b) a synthetic RNA encoding agene-editing protein capable of creating a double strand break in FXNB(SEQ ID NO: 583), wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity. Invarious embodiments, the treatment comprises administering to a subjectin need thereof an effective amount of (a) the synthetic RNA encodingthe gene-editing protein capable of creating a double strand break inFXNA (SEQ ID NO: 582), and (b) the synthetic RNA encoding thegene-editing protein capable of creating a double strand break in FXNB(SEQ ID NO: 583).

In various embodiments, the administration is directed to the heart.

In various embodiments, the administration is via a catheter.

In various embodiments, the treatment ameliorates one or more of muscleweakness, loss of coordination, vision impairment, hearing impairment,slurred speech, scoliosis, pes cavus deformity of the foot, diabetes,and heart disorders, selected from one or more atrial fibrillation,tachycardia and hypertrophic cardiomyopathy.

In various embodiments, the gene-editing protein is selected from aCRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) aDNA-binding domain and (b) a nuclease domain. The DNA-binding domaincomprises a plurality of repeat sequences and at least one of the repeatsequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ IDNO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, Dor E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S,or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises acatalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming adimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalyticdomain of a protein comprising the amino acid sequence of SEQ ID NO:632.

In some aspects, the present invention relates to a method for reducinginflammation. The method comprises a step of administering an effectiveamount of a synthetic RNA encoding superoxide dismutase 3 (SOD3) orIF_(κ)B, wherein the synthetic RNA comprises one or more non-canonicalnucleotides that avoid substantial cellular toxicity to a subject inneed thereof.

In various embodiments, the inflammation is associated with a lungdisease or disorder.

In various embodiments, the lung disease or disorder is selected fromAsbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, ChronicObstructive Pulmonary Disease (COPD), Common Cold, Croup, CysticFibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis, Influenza, LungCancer, Pandemic Flu, Pertussis, Pleurisy, Pneumonia, PulmonaryEmbolism, Pulmonary Hypertension, Respiratory Syncytial Virus (RSV),Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome(SIDS), and Tuberculosis.

In various embodiments, the inflammation is associated with a lunginfection.

In various embodiments, the lung infection is cause by a bacterium, afungus, a protozoa, a multi-cellular organism, a particulate, or avirus.

In various embodiments, the synthetic RNA is administered by inhalation.

In various embodiments, the inflammation is associated with sepsis.

In some aspects, the present invention relates to a method for treatingalpha-1 antitrypsin (A1AT) deficiency. The method comprises a step ofadministering to a subject in need thereof an effective amount of (a) asynthetic RNA encoding a gene-editing protein capable of creating adouble strand break in A1AT_A (SEQ ID NO: 584) and/or (b) a syntheticRNA encoding a gene-editing protein capable of creating a double strandbreak in A1AT_B (SEQ ID NO: 585), wherein the synthetic RNA comprisesone or more non-canonical nucleotides that avoid substantial cellulartoxicity. In various embodiments, the treatment comprises administeringto a subject in need thereof an effective amount of (a) the syntheticRNA encoding the gene-editing protein capable of creating a doublestrand break in A1AT_A (SEQ ID NO: 584) and (b) the synthetic RNAencoding the a gene-editing protein capable of creating a double strandbreak in A1AT_B (SEQ ID NO: 585).

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the treatment corrects the Z mutation in thesubject's liver cells.

In various embodiments, the treatment reduces polymerized Z proteinaccumulation in the subject's liver cells.

In various embodiments, the treatment increases secretion and/or serumlevels of functional A1AT.

In various embodiments, the treatment ameliorates one or more of chroniccough, emphysema, COPD, liver failure, hepatitis, hepatomegaly,jaundice, and cirrhosis.

In various embodiments, the gene-editing protein is selected from aCRISPR/Cas9, TALEN, and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) aDNA-binding domain and (b) a nuclease domain. The DNA-binding domaincomprises a plurality of repeat sequences and at least one of the repeatsequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ IDNO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, Dor E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S,or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises acatalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming adimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalyticdomain of a protein comprising the amino acid sequence of SEQ ID NO:632.

In some aspects, the present invention relates to a method for treatingalpha-1 antitrypsin (A1AT) deficiency. The method comprises a step ofadministering an effective amount of a synthetic RNA encoding A1AT to asubject, wherein the synthetic RNA comprises one or more non-canonicalnucleotides that avoid substantial cellular toxicity.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the treatment corrects the Z mutation in thesubject's liver cells.

In various embodiments, the treatment reduces polymerized Z proteinaccumulation in the subject's liver cells.

In various embodiments, the treatment increases secretion and/or serumlevels of functional A1AT.

In various embodiments, the treatment ameliorates one or more of thesubject's symptoms.

In some aspects, the present invention relates to a method for reversingan alpha-1 antitrypsin (A1AT) deficiency in a cell. The method comprisessteps of (a) obtaining a cell comprising a defective A1AT gene and (b)contacting the cell with a synthetic RNA encoding A1AT, wherein thesynthetic RNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.

In various embodiments, the method further comprises steps of (c)obtaining the cell contacted in step (b); and (d) administering the cellto a subject in need thereof.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In some aspects, the present invention relates to a method for reversingan alpha-1 antitrypsin (A1AT) deficiency in a cell. The method comprisessteps of (a) obtaining a cell comprising a defective A1AT gene and (b)contacting the cell with one or both of a synthetic RNA encoding agene-editing protein capable of creating a double strand break in A1AT_A(SEQ ID NO: 584) and a synthetic RNA encoding a gene-editing proteincapable of creating a double strand break in A1AT_B (SEQ ID NO: 585),wherein the synthetic RNA comprises one or more non-canonicalnucleotides that avoid substantial cellular toxicity.

In various embodiments, the method further comprises steps of (c)obtaining the cell contacted in step (b); and (d) administering the cellto a subject in need thereof.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the gene-editing protein is selected from aCRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) aDNA-binding domain and (b) a nuclease domain.

The DNA-binding domain comprises a plurality of repeat sequences and atleast one of the repeat sequences comprises the amino acid sequence:LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acidslong, wherein: “v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y”is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domaincomprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming adimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalyticdomain of a protein comprising the amino acid sequence of SEQ ID NO:632.

In some aspects, the present invention relates to a method formodulating BMP7. The method comprises a step of administering aneffective amount of a synthetic RNA encoding BMP7 to a subject, whereinthe synthetic RNA comprises one or more non-canonical nucleotides thatavoid substantial cellular toxicity.

In various embodiments, the modulating results in an increase in thequantity of BMP7 in the subject.

In various embodiments, the modulating results in a decrease in thequantity of BMP7 in the subject.

In various embodiments, the modulating results in an increase inintracellular alkaline phosphatase activity.

In various embodiments, the synthetic RNA encoding BMP7 comprises asequence encoding the BMP7 signal peptide.

In various embodiments, the BMP7 signal peptide comprises the sequenceof SEQ ID NO: 597.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the administration treats diabetic nephropathy.

In various embodiments, the administration treats liver fibrosis.

In various embodiments, the administration is directed to the kidney.

In various embodiments, the administration is intravenous orintradermal.

In various embodiments, the administration treats kidney disease.

In various embodiments, the kidney disease is diabetic nephropathy.

In various embodiments, the modulating results in reduced levels ofurine albumin in the subject.

In some aspects, the present invention relates to a method formodulating an immune checkpoint molecule. The method comprises a step ofadministering an effective amount of a synthetic RNA encoding the immunecheckpoint molecule to a subject, wherein the synthetic RNA comprisesone or more non-canonical nucleotides that avoid substantial cellulartoxicity.

In some aspects, the present invention relates to a method for treatinga subject comprising administering a step of a synthetic RNA encoding agene-editing protein targeting an immune checkpoint molecule gene.

In some aspects, the present invention relates to a method for treatinga subject comprising steps of (a) obtaining a cell comprising an immunecheckpoint molecule gene and (b) contacting the cell with a syntheticRNA encoding a gene-editing protein capable of creating a double strandbreak in the immune checkpoint molecule gene, wherein the synthetic RNAcomprises one or more non-canonical nucleotides that avoid substantialcellular toxicity.

In various embodiments, the method further comprises steps of (c)obtaining the cell contacted in step (b); and (d) administering the cellto a subject in need thereof.

In various embodiments, the gene-editing protein is selected from aCRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) aDNA-binding domain and (b) a nuclease domain. The DNA-binding domaincomprises a plurality of repeat sequences and at least one of the repeatsequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ IDNO: 629) and is between 36 and 39 amino acids long, wherein:

“v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N,G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises acatalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming adimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalyticdomain of a protein comprising the amino acid sequence of SEQ ID NO:632.

In various embodiments, the immune checkpoint molecule is selected fromPD-1, PD-L1, PD-L2, CTLA-4, ICOS, LAG3, OX40, OX40L, and TIM3.

In various embodiments, the immune checkpoint molecule is PD-1.

In various embodiments, the administering stimulates or enhances animmune response in the subject.

In various embodiments, the administering inhibits or reduces an immuneresponse in the subject.

In various embodiments, the subject is afflicted with a cancer.

In various embodiments, the subject is afflicted with an autoimmunedisease.

In various embodiments, the administration is intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,intraportal, rectally, by inhalation, or topically.

In various embodiments, the modulating results in an increase in thequantity of PD-1 in the subject.

In various embodiments, the modulating results in a decrease in thequantity of PD-1 in the subject.

In some aspects, the present invention relates to a method for treatinga cancer comprising steps of (a) isolating an cell from a subject, thecell being an immune cell or hematopoietic cell; (b) contacting theisolated cell with an effective amount of a synthetic RNA encoding achimeric antigen receptor (CAR), wherein the synthetic RNA comprises oneor more non-canonical nucleotides that avoid substantial cellulartoxicity; and (c) administering the cell to the subject.

In various embodiments, the immune cell is a T cell.

In some aspects, the present invention relates to a method for making achimeric antigen receptor (CAR) T cell comprising steps of (a) obtaininga cell from a subject and (b) contacting the cell with a synthetic RNAencoding a gene-editing protein capable of creating a double strandbreak to yield a safe harbor locus for CAR insertion, wherein thesynthetic RNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.

In various embodiments, the wherein the gene-editing protein is selectedfrom a CRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) aDNA-binding domain and (b) a nuclease domain. The DNA-binding domaincomprises a plurality of repeat sequences and at least one of the repeatsequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ IDNO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, Dor E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S,or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises acatalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming adimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalyticdomain of a protein comprising the amino acid sequence of SEQ ID NO:632.

In some aspects, the present invention relates to a method forincreasing the persistence of a chimeric antigen receptor (CAR) T cell,comprising a step of contacting the chimeric antigen receptor (CAR) Tcell with a synthetic RNA encoding a encoding a telomerase, wherein thesynthetic RNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.

In some aspects, the present invention relates to a method for treatinga skin wound, comprising a step of administering an effective amount ofa synthetic RNA encoding interleukin 22 (IL22) to a subject, wherein thesynthetic RNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.

In various embodiments, the administration is directed to a populationof cells of the integumentary system.

In various embodiments, the population of cells comprises one or more ofcells of the epidermis, cells of the basement membrane, cells of thedermis, and/or cells of the subcutis.

In various embodiments, the population of cells is cells of theepidermis, and comprises one more of cells of the stratum corneum,stratum lucidum, stratum granulosum, stratum spinosum, and/or stratumgerminativum.

In various embodiments, the population of cells is cells of the dermis,and comprises one or more of cells from the papillary region and thereticular region.

In various embodiments, the administration is by subcutaneous injection,intradermal injection, subdermal injection, intramuscular injection, ortopical administration.

In various embodiments, the administration is intradermal injection toone or more of the dermis or epidermis.

In various embodiments, the an effective amount is from about 10 ng toabout 5000 ng per treatment area of about 10 cm² or less, or about 5 cm²or less, or about 1 cm² or less, or about 0.5 cm² or less, or about 0.2cm² or less.

In various embodiments, about 10 ng, or about 20 ng, or about 50 ng, orabout 100 ng, or about 200 ng, or about 300 ng, or about 400 ng, orabout 500 ng, or about 600 ng, or about 700 ng, or about 800 ng, orabout 900 ng, or about 1000 ng, or about 1100 ng, or about 1200 ng, orabout 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600 ng, orabout 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000 ng, orabout 3000 ng, or about 4000 ng, or about 5000 ng of the synthetic RNAis administered per treatment area of about 10 cm² or less, or about 5cm² or less, or about 1 cm² or less, or about 0.5 cm² or less, or about0.2 cm² or less.

In various embodiments, the modulating results in an increase in thequantity of IL22 in the subject.

In various embodiments, the modulating results in a decrease in thequantity of IL22 in the subject.

In various embodiments, the effective amount of synthetic RNA isadministered using an array of needles covering an affected area of thesubject.

In various embodiments, the treatment area is mechanically massagedafter administration.

In various embodiments, the treatment area is exposed to electric pulsesafter administration.

In various embodiments, the electric pulses are between about 10V andabout 200V for from about 50 microseconds to about 1 second.

In various embodiments, the electric pulses are generated around thetreatment area by a multielectrode array.

In some aspects, the present invention relates to a method for treatinga metabolic disorder, comprising a step of administering a synthetic RNAencoding a gene-editing protein targeting a defective gene, wherein theadministration is by intraportal injection.

In some aspects, the present invention relates to a method for treatinga metabolic disorder comprising a step of (a) obtaining a cellcomprising a defective gene and (b) contacting the cell with a syntheticRNA encoding a gene-editing protein capable of creating a double strandbreak in the defective gene, wherein the synthetic RNA comprises one ormore non-canonical nucleotides that avoid substantial cellular toxicity.In various embodiments, the method further comprises steps of (c)obtaining the cell contacted in step (b); and (d) administering the cellto a subject in need thereof by intraportal injection.

In various embodiments, the gene-editing protein is selected from aCRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) aDNA-binding domain and (b) a nuclease domain. The DNA-binding domaincomprises a plurality of repeat sequences and at least one of the repeatsequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ IDNO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, Dor E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S,or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises acatalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming adimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalyticdomain of a protein comprising the amino acid sequence of SEQ ID NO:632.

In various embodiments, the metabolic disorder is selected from adisorder of carbohydrate metabolism, a disorder of amino acidmetabolism, a disorder of the urea cycle, a disorder of fatty acidmetabolism, a disorder of porphyrin metabolism, a disorder of lysosomalstorage, a disorder of peroxisome biogenesis, and a disorder of purineor pyrimidine metabolism.

In various embodiments, the metabolic disorder is a disorder ofcarbohydrate metabolism and wherein the disease is galactosemia and thedefective gene is optionally GALT, GALK1, or GALE; wherein the diseaseis essential fructosuria and the defective gene is optionally KHK;wherein the disease is Hereditary fructose intolerance and the defectivegene is optionally ALDOB; wherein the disease is glycogen storagedisease type I and the defective gene is optionally G6PC, SLC37A4, orSLC17A3; wherein the disease is glycogen storage disease type II and thedefective gene is optionally GAA; wherein the disease is glycogenstorage disease type III and the defective gene is optionally AGL;wherein the disease is glycogen storage disease type IV and thedefective gene is optionally GBE1; wherein the disease is glycogenstorage disease type V and the defective gene is optionally PYGM;wherein the disease is glycogen storage disease type VI and thedefective gene is optionally PYGL; wherein the disease is glycogenstorage disease type VII and the defective gene is optionally PYGM;wherein the disease is glycogen storage disease type IX and thedefective gene is optionally PHKA1, PHKA2, PHKB, PHKG1, or PHKG2;wherein the disease is glycogen storage disease type XI and thedefective gene is optionally SLC2A2; wherein the disease is glycogenstorage disease type XII and the defective gene is optionally ALDOA;wherein the disease is glycogen storage disease type XIII and thedefective gene is optionally ENO1, ENO2, or ENO3; wherein the disease isglycogen storage disease type 0 and the defective gene is optionallyGYS1 or GYS2; wherein the disease is pyruvate carboxylase deficiency andthe defective gene is optionally PC; wherein the disease is pyruvatekinase deficiency and the defective gene is optionally PKLR; wherein thedisease is transaldolase deficiency and the defective gene is optionallyTALDO1; wherein the disease is triosephosphate isomerase deficiency andthe defective gene is optionally TPI1; wherein the disease is fructosebisphosphatase deficiency and the defective gene is optionally FBP1;wherein the disease is hyperoxaluria and the defective gene isoptionally AGXT or GRHPR; wherein the disease is hexokinase deficiencyand the defective gene is optionally HK1; wherein the disease isglucose-galactose malabsorption and the defective gene is optionallySLC5A1; or wherein the disease is glucose-6-phosphate dehydrogenasedeficiency and the defective gene is optionally G6PD.

In various embodiments, the metabolic disorder is a disorder of aminoacid metabolism wherein the disease is alkaptonuria and the defectivegene is optionally HGD; wherein the disease is aspartylglucosaminuriaand the defective gene is optionally AGA; wherein the disease ismethylmalonic acidemia and the defective gene is optionally MUT, MCEE,MMAA, MMAB, MMACHC, MMADHC, or LMBRD1; wherein the disease is maplesyrup urine disease and the defective gene is optionally BCKDHA, BCKDHB,DBT, or DLD; wherein the disease is homocystinuria and the defectivegene is optionally CBS; wherein the disease is tyrosinemia and thedefective gene is optionally FAH, TAT, or HPD; wherein the disease istrimethylaminuria and the defective gene is optionally FMO3; wherein thedisease is Hartnup disease and the defective gene is optionally SLC6A19;wherein the disease is biotinidase deficiency and the defective gene isoptionally BTD; wherein the disease is ornithine carbamoyltransferasedeficiency and the defective gene is optionally OTC; wherein the diseaseis carbamoyl-phosphate synthase I deficiency disease and the defectivegene is optionally CPS1; wherein the disease is citrullinemia and thedefective gene is optionally ASS or SLC25A13; wherein the disease ishyperargininemia and the defective gene is optionally ARG1; wherein thedisease is hyperhomocysteinemia and the defective gene is optionallyMTHFR; wherein the disease is hypermethioninemia and the defective geneis optionally MAT1A, GNMT, or AHCY; wherein the disease ishyperlysinemias and the defective gene is optionally AASS; wherein thedisease is nonketotic hyperglycinemia and the defective gene isoptionally GLDC, AMT, or GCSH; wherein the disease is Propionic acidemiaand the defective gene is optionally PCCA or PCCB; wherein the diseaseis hyperprolinemia and the defective gene is optionally ALDH4A1 orPRODH; wherein the disease is cystinuria and the defective gene isoptionally SLC3A1 or SLC7A9; wherein the disease is dicarboxylicaminoaciduria and the defective gene is optionally SLC1A1; wherein thedisease is glutaric acidemia type 2 and the defective gene is optionallyETFA, ETFB, or ETFDH; wherein the disease is isovaleric acidemia and thedefective gene is optionally IVD; or wherein the disease is2-hydroxyglutaric aciduria and the defective gene is optionally L2HGDHor D2HGDH.

In various embodiments, the metabolic disorder is a disorder of the ureacycle wherein the disease is N-acetylglutamate synthase deficiency andthe defective gene is optionally NAGS; wherein the disease isargininosuccinic aciduria and the defective gene is optionally ASL; orwherein the disease is argininemia and the defective gene is optionallyARG1.

In various embodiments, the metabolic disorder is a disorder of fattyacid metabolism wherein the disease is very long-chain acyl-coenzyme Adehydrogenase deficiency and the defective gene is optionally ACADVL;wherein the disease is long-chain 3-hydroxyacyl-coenzyme A dehydrogenasedeficiency and the defective gene is optionally HADHA; wherein thedisease is medium-chain acyl-coenzyme A dehydrogenase deficiency and thedefective gene is optionally ACADM; wherein the disease is short-chainacyl-coenzyme A dehydrogenase deficiency and the defective gene isoptionally ACADS; wherein the disease is 3-hydroxyacyl-coenzyme Adehydrogenase deficiency and the defective gene is optionally HADH;wherein the disease is 2,4 dienoyl-CoA reductase deficiency and thedefective gene is optionally NADK2; wherein the disease is3-hydroxy-3-methylglutaryl-CoA lyase deficiency and the defective geneis optionally HMGCL; wherein the disease is malonyl-CoA decarboxylasedeficiency and the defective gene is optionally MLYCD; wherein thedisease is systemic primary carnitine deficiency and the defective geneis optionally SLC22A5; wherein the disease is carnitine-acylcarnitinetranslocase deficiency and the defective gene is optionally SLC25A20;wherein the disease is carnitine palmitoyltransferase I deficiency andthe defective gene is optionally CPT1A; wherein the disease is carnitinepalmitoyltransferase II deficiency and the defective gene is optionallyCPT2; wherein the disease is lysosomal acid lipase deficiency and thedefective gene is optionally LIPA; or wherein the disease is Gaucher'sdisease and the defective gene is optionally GBA.

In various embodiments, the metabolic disorder is a disorder ofporphyrin metabolism wherein the disease is acute intermittent porphyriaand the defective gene is optionally HMBS; wherein the disease isGunther disease and the defective gene is optionally UROS; wherein thedisease is porphyria cutanea tarda and the defective gene is optionallyUROD; wherein the disease is hepatoerythropoietic porphyria and thedefective gene is optionally UROD; wherein the disease is hereditarycoproporphyria and the defective gene is optionally CPDX; wherein thedisease is variegate porphyria and the defective gene is optionallyPPDX; wherein the disease is erythropoietic protoporphyria and thedefective gene is optionally FECH; or wherein the disease isaminolevulinic acid dehydratase deficiency porphyria and the defectivegene is optionally ALAD.

In various embodiments, the metabolic disorder is a disorder oflysosomal storage wherein the disease is Farber disease and thedefective gene is optionally ASAH1; wherein the disease is Krabbedisease and the defective gene is optionally GALC; wherein the diseaseis galactosialidosis and the defective gene is optionally CTSA; whereinthe disease is fabry disease and the defective gene is optionally GLA;wherein the disease is Schindler disease and the defective gene isoptionally NAGA; wherein the disease is GM1 gangliosidosis and thedefective gene is optionally GLB1; wherein the disease is Tay-Sachsdisease and the defective gene is optionally HEXA; wherein the diseaseis Sandhoff disease and the defective gene is optionally HEXB; whereinthe disease is GM2-gangliosidosis, AB variant and the defective gene isoptionally GM2A; wherein the disease is Niemann-Pick disease and thedefective gene is optionally SMPD1, NPC1, or NPC2; wherein the diseaseis metachromatic leukodystrophy and the defective gene is optionallyARSA or PSAP; wherein the disease is multiple sulfatase deficiency andthe defective gene is optionally SUMF1; wherein the disease is Hurlersyndrome and the defective gene is optionally IDUA; wherein the diseaseis Hunter syndrome and the defective gene is optionally IDS; wherein thedisease is Sanfilippo syndrome and the defective gene is optionallySGSH, NAGLU, HGSNAT, or GNS; wherein the disease is Morquio syndrome andthe defective gene is optionally GALNS or GLB1; wherein the disease isMaroteaux-Lamy syndrome and the defective gene is optionally ARSB;wherein the disease is Sly syndrome and the defective gene is optionallyGUSB; wherein the disease is sialidosis and the defective gene isoptionally NEU1, NEU2, NEU3, or NEU4; wherein the disease is I-celldisease and the defective gene is optionally GNPTAB or GNPTG; whereinthe disease is mucolipidosis type IV and the defective gene isoptionally MCOLN1; wherein the disease is infantile neuronal ceroidlipofuscinosis and the defective gene is optionally PPT1 or PPT2;wherein the disease is Jansky-Bielschowsky disease and the defectivegene is optionally TPP1; wherein the disease is Batten disease and thedefective gene is optionally CLN1, CLN2, CLN3, CLN5, CLN6, MFSD8, CLN8,or CTSD; wherein the disease is Kufs disease, Type A and the defectivegene is optionally CLN6 or PPT1; wherein the disease is Kufs disease,Type B and the defective gene is optionally DNAJC5 or CTSF; wherein thedisease is alpha-mannosidosis and the defective gene is optionallyMAN2B1, MAN2B2, or MAN2C1; wherein the disease is beta-mannosidosis andthe defective gene is optionally MANBA; wherein the disease isfucosidosis and the defective gene is optionally FUCA1; wherein thedisease is cystinosis and the defective gene is optionally CTNS; whereinthe disease is pycnodysostosis and the defective gene is optionallyCTSK; wherein the disease is Salla disease and the defective gene isoptionally SLC17A5; wherein the disease is Infantile free sialic acidstorage disease and the defective gene is optionally SLC17A5; or whereinthe disease is Danon disease and the defective gene is optionally LAMP2.

In various embodiments, the metabolic disorder is a disorder ofperoxisome biogenesis wherein the disease is Zellweger syndrome and thedefective gene is optionally PEX1, PEX2, PEX3, PEX5, PEX6, PEX12, PEX14,or PEX26; wherein the disease is Infantile Refsum disease and thedefective gene is optionally PEX1, PEX2, or PEX26; wherein the diseaseis neonatal adrenoleukodystrophy and the defective gene is optionallyPEX5, PEX1, PEX10, PEX13, or PEX26; wherein the disease is RCDP Type 1and the defective gene is optionally PEX7; wherein the disease ispipecolic acidemia and the defective gene is optionally PAHX; whereinthe disease is acatalasia and the defective gene is optionally CAT;wherein the disease is hyperoxaluria type 1 and the defective gene isoptionally AGXT; wherein the disease is Acyl-CoA oxidase deficiency andthe defective gene is optionally ACOX1; wherein the disease isD-bifunctional protein deficiency and the defective gene is optionallyHSD17B4; wherein the disease is dihydroxyacetonephosphateacyltransferase deficiency and the defective gene is optionally GNPAT;wherein the disease is X-linked adrenoleukodystrophy and the defectivegene is optionally ABCD1; wherein the disease is α-methylacyl-CoAracemase deficiency and the defective gene is optionally AMACR; whereinthe disease is RCDP Type 2 and the defective gene is optionally DHAPAT;wherein the disease is RCDP Type 3 and the defective gene is optionallyAGPS; wherein the disease is adult refsum disease-1 and the defectivegene is optionally PHYH; or wherein the disease is mulibrey nanism andthe defective gene is optionally TRIM37.

In various embodiments, the metabolic disorder is a disorder of purineor pyrimidine metabolism wherein the disease is Lesch-Nyhan syndrome andthe defective gene is optionally HPRT; wherein the disease is adeninephosphoribosyltransferase deficiency and the defective gene isoptionally APRT; wherein the disease is adenosine deaminase deficiencyand the defective gene is optionally ADA; wherein the disease isAdenosine monophosphate deaminase deficiency type 1 and the defectivegene is optionally AMPD1; wherein the disease is adenylosuccinate lyasedeficiency and the defective gene is optionally ADSL; wherein thedisease is dihydropyrimidine dehydrogenase deficiency and the defectivegene is optionally DPYD; wherein the disease is Miller syndrome and thedefective gene is optionally DHODH; wherein the disease is oroticaciduria and the defective gene is optionally UMPS; wherein the diseaseis purine nucleoside phosphorylase deficiency and the defective gene isoptionally PNP; or wherein the disease is xanthinuria and the defectivegene is optionally XDH, MOCS1, or MOCS2, GEPH.

In some aspects, the present invention relates to a method formodulating transthyretin (TTR). The method comprising a step ofadministering an effective amount of a synthetic RNA encoding TTR to asubject, wherein the synthetic RNA comprises one or more non-canonicalnucleotides that avoid substantial cellular toxicity.

In various embodiments, the modulating results in an increase in thequantity of TTR in the subject.

In various embodiments, the modulating results in a decrease in thequantity of TTR in the subject.

In various embodiments, the modulating results in treatment of one ormore of an amyloid disease, senile systemic amyloidosis (SSA), familialamyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC).

In various embodiments, the non-canonical nucleotides have one or moresubstitutions at positions selected from the 2C, 4C, and 5C positionsfor a pyrimidine, or selected from the 6C, 7N and 8C positions for apurine.

In various embodiments, the non-canonical nucleotides comprise one ormore of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine,5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine,5-carboxyuridine, 5-formyluridine, 5-methoxyuridine,5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridine, optionally at anamount of at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90%, or 100% of the non-canonical nucleotides.

In various embodiments, the at least about 50% of cytidine residues arenon-canonical nucleotides, and which are selected from5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

In various embodiments, the at least about 75% or at least about 90% ofcytidine residues are non-canonical nucleotides, and the non-canonicalnucleotides are selected from 5-hydroxycytidine, 5-methylcytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and5-methoxycytidine.

In various embodiments, the at least about 20% of uridine, or at leastabout 40%, or at least about 50%, or at least about 75%, or at aboutleast 90% of uridine residues are non-canonical nucleotides, and thenon-canonical are selected from pseudouridine, 5-hydroxyuridine,5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine,5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine,5-methylpseudouridine, 5-hydroxymethylpseudouridine,5-carboxypseudouridine, 5-formylpseudouridine, and5-methoxypseudouridine.

In various embodiments, the at least about 40%, or at least about 50%,or at least about 75%, or at about least 90% of uridine residues arenon-canonical nucleotides, and the non-canonical nucleotides areselected from pseudouridine, 5-hydroxyuridine, 5-methyluridine,5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine,5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridine.

In various embodiments, the at least about 10% of guanine residues arenon-canonical nucleotides, and the non-canonical nucleotide isoptionally 7-deazaguanosine.

In various embodiments, the synthetic RNA contains no more than about50% 7-deazaguanosine in place of guanosine residues.

In various embodiments, the synthetic RNA does not contain non-canonicalnucleotides in place of adenosine residues.

In various embodiments, the synthetic RNA comprises a 5′ cap structure.

In various embodiments, the synthetic RNA 5′-UTR comprises a Kozakconsensus sequence.

In various embodiments, the synthetic RNA 5′-UTR comprises a sequencethat increases RNA stability in vivo, and the 5′-UTR may comprise analpha-globin or beta-globin 5′-UTR.

In various embodiments, the synthetic RNA 3′-UTR comprises a sequencethat increases RNA stability in vivo, and the 3′-UTR may comprise analpha-globin or beta-globin 3′-UTR.

In various embodiments, the synthetic RNA comprises a 3′ poly(A) tail.

In various embodiments, the synthetic RNA 3′ poly(A) tail is from about20 nucleotides to about 250 nucleotides in length.

In various embodiments, the synthetic RNA is from about 200 nucleotidesto about 5000 nucleotides in length.

In various embodiments, the synthetic RNA is from about 500 to about2000 nucleotides in length, or about 500 to about 1500 nucleotides inlength, or about 500 to about 1000 nucleotides in length.

In various embodiments, the synthetic RNA is prepared by in vitrotranscription.

In various embodiments, the effective amount of the synthetic RNA isadministered as one or more injections containing about 10 ng to about5000 ng of RNA.

In various embodiments, the effective amount of the synthetic RNA isadministered as one or more injections each containing no more thanabout 10 ng, or no more than about 20 ng, or no more than about 50 ng,or no more than about 100 ng, or no more than about 200 ng, or no morethan about 300 ng, or no more than about 400 ng, or no more than about500 ng, or no more than about 600 ng, or no more than about 700 ng, orno more than about 800 ng, or no more than about 900 ng, or no more thanabout 1000 ng, or no more than about 1100 ng, or no more than about 1200ng, or no more than about 1300 ng, or no more than about 1400 ng, or nomore than about 1500 ng, or no more than about 1600 ng, or no more thanabout 1700 ng, or no more than about 1800 ng, or no more than about 1900ng, or no more than about 2000 ng, or no more than about 3000 ng, or nomore than about 4000 ng, or no more than about 5000 ng.

In various embodiments, the effective amount of the synthetic RNA isadministered as one or more injections each containing about 10 ng, orabout 20 ng, or about 50 ng, or about 100 ng, or about 200 ng, or about300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000ng.

In various embodiments, the effective amount of the synthetic RNAcomprises one or more lipids to enhance uptake of RNA by cells.

In various embodiments, the effective amount of the synthetic RNAcomprises a cationic liposome formulation and the lipids are optionallyselected from Table 1.

In various embodiments, the subject is a human.

In various embodiments, the effective amount of the synthetic RNA isadministered about weekly, for at least 2 weeks.

In various embodiments, the effective amount of the synthetic RNA isadministered about every other week for at least one month.

In various embodiments, the effective amount of the synthetic RNA isadministered monthly or about every other month.

In various embodiments, the effective amount of the synthetic RNA isadministered for at least two months, or at least 4 months, or at least6 months, or at least 9 months, or at least one year.

In some aspects, the present invention relates to a compositioncomprising an effective amount of the synthetic RNA used in anyherein-disclosed aspect or embodiment.

In some aspects, the present invention relates to a pharmaceuticalcomposition, comprising the composition of any herein-disclosed aspector embodiment and a pharmaceutically acceptable excipient.

In some aspects, the present invention relates to the use of acomposition or pharmaceutical composition of any herein-disclosed aspector embodiment in the treatment of a disease or disorder describedherein.

In some aspects, the present invention relates to the use of acomposition or pharmaceutical composition of any herein-disclosed aspector embodiment in the manufacture of a medicament for the treatment of adisease or disorder described herein.

In various aspects, the present invention is based on the surprisingdiscovery of safe and effective doses and administration parameters fornucleic acid drugs in human subjects. In some aspects, there is provideda method for delivering a nucleic acid drug, comprising administering aneffective dose of a nucleic acid drug to a human subject in needthereof. In various embodiments, the nucleic acid drug comprises a RNAcomprising one or more non-canonical nucleotides (a/k/a “modified RNA”).In other embodiments, the effective dose is an amount sufficient tosubstantially increase an amount of a protein encoded by the nucleicacid drug in the human subject and/or substantially avoid an immunereaction in a human subject, wherein the immune reaction is optionallymediated by the innate immune system.

In some aspects, there is provided a method for expressing a protein ofinterest in a population of cells in a mammalian subject, comprisingadministering a non-viral transfection composition comprising aneffective dose of a RNA encoding the protein of interest to said cells,where the transfection composition is administered in an amount thatallows for expression of said protein in said cells for at least aboutsix hours to about five days without substantial cellular toxicity. Insome embodiments, the RNA contains one or more non-canonical nucleotidesthat avoid substantial cellular toxicity.

In some embodiments, the effective dose is about 100 ng to about 5000 ng(e.g., about, or no more than about, 100 ng, or 200 ng, or 300 ng, or400 ng, or 500 ng, or 600 ng, or 700 ng, or 800 ng, or 900 ng, or 1000ng, or 1100 ng, or 1200 ng, or 1300 ng, or 1400 ng, or 1500 ng, or 1600ng, or 1700 ng, or 1800 ng, or 1900 ng, or 2000 ng, or 3000 ng, or 4000ng, or 5000 ng). In other embodiments, the effective dose is less thanabout 100 ng. In certain embodiments, the effective dose is about 10 ngto about 100 ng (e.g., about, or no more than about, 10 ng, or 20 ng, or30 ng, or 40 ng, or 50 ng, or 60 ng, or 70 ng, or 80 ng, or 90 ng, or100 ng).

In some embodiments, the effective dose is about 1.4 ng/kg to about 30ng/kg (e.g., about, or no more than about, 1.4 ng/kg, or 2.5 ng/kg, or 5ng/kg, or 10 ng/kg, or 15 ng/kg, or 20 ng/kg, or 25 ng/kg, or 30 ng/kg.In other embodiments, the effective dose is less than about 1.5 ng/kg.In certain embodiments, the effective dose is about 0.14 ng/kg to about1.4 ng/kg (e.g., about, or no more than about, 0.14 ng/kg, or 0.25ng/kg, or 0.5 ng/kg, or 0.75 ng/kg, or 1 ng/kg, or 1.25 ng/kg, or 1.4ng/kg).

In some embodiments, the effective dose is about 350 ng/cm² to about7000 ng/cm² (e.g., about, or no more than about, 350 ng/cm², or 500ng/cm², or 750 ng/cm², or 1000 ng/cm², or 2000 ng/cm², or 3000 ng/cm²,or 4000 ng/cm², or 5000 ng/cm², or 6000 ng/cm², or 7000 ng/cm²). Inother embodiments, the effective dose is less than about 350 ng/cm². Incertain embodiments, the effective dose is about 35 ng/cm² to about 350ng/cm² (e.g., about, or no more than about, 35 ng/cm², or 50 ng/cm², or75 ng/cm², or 100 ng/cm², or 150 ng/cm², or 200 ng/cm², or 250 ng/cm²,or 300 ng/cm², or 350 ng/cm².

In some embodiments, the effective dose is about 0.28 picomoles to about5.7 picomoles (e.g., about, or no more than about, 0.28 picomoles, or0.5 picomoles, or 0.75 picomoles, or 1 picomole, or 2 picomoles, or 3picomoles, or 4 picomoles, or 5 picomoles, or 5.7 picomoles). In otherembodiments, the effective dose is less than about 0.28 picomoles. Incertain embodiments, the effective dose is about 0.028 picomoles toabout 0.28 picomoles (e.g., about, or no more than about, 0.028picomoles, or 0.05 picomoles, or 0.075 picomoles, or 0.1 picomoles, or0.15 picomoles, or 0.2 picomoles, or 0.25 picomoles, or 0.28 picomoles).

In some embodiments, the effective dose is about 0.004 picomoles/kg toabout 0.082 picomoles/kg (e.g., about, or no more than about, 0.004picomoles/kg, or 0.01 picomoles/kg, or 0.02 picomoles/kg, or 0.03picomoles/kg, or 0.04 picomoles/kg, or 0.05 picomoles/kg, or 0.06picomoles/kg, or 0.07 picomoles/kg, or 0.08 picomoles/kg, or 0.082picomoles/kg). In other embodiments, the effective dose is less thanabout 0.004 picomoles/kg. In certain embodiments, the effective dose isabout 0.0004 picomoles/kg to about 0.004 picomoles/kg (e.g., about, orno more than about, 0.0004 picomoles/kg, or 0.001 picomoles/kg, or 0.002picomoles/kg, or 0.003 picomoles/kg, or 0.004 picomoles/kg).

In some embodiments, the effective dose is about 1 picomole/cm² to about20 picomoles/cm² (e.g., about, or no more than about, 1 picomole/cm², or2 picomoles/cm², or 3 picomoles/cm², or 4 picomoles/cm², or 5picomoles/cm², or 6 picomoles/cm², or 7 picomoles/cm², or 8picomoles/cm², or 9 picomoles/cm², or 10 picomoles/cm², or 12picomoles/cm², or 14 picomoles/cm², or 16 picomoles/cm², or 18picomoles/cm², or 20 picomoles/cm²). In other embodiments, the effectivedose is less than about 1 picomole/cm². In certain embodiments, theeffective dose is about 0.1 picomoles/cm² to about 1 picomole/cm² (e.g.,about, or no more than about, 0.1 picomoles/cm², or 0.2 picomoles/cm²,or 0.3 picomoles/cm², or 0.4 picomoles/cm², or 0.5 picomoles/cm², or 0.6picomoles/cm², or 0.7 picomoles/cm², or 0.8 picomoles/cm², or 0.9picomoles/cm², or 1 picomole/cm²).

In various embodiments, the nucleic acid drug is administered in apharmaceutically acceptable formulation, including a formulationsuitable for one or more of injection (e.g. subcutaneous injection,intradermal injection (including to the dermis or epidermis), subdermalinjection, intramuscular injection, intraocular injection, intravitrealinjection, intra-articular injection, intracardiac injection,intravenous injection, epidural injection, intrathecal injection,intraportal injection, intratumoral injection) and topicaladministration and/or for administration to the integumentary system(e.g. to one or more of the epidermis (optionally selected from thestratum corneum, stratum lucidum, stratum granulosum, stratum spinosum,and stratum germinativum), basement membrane, dermis (optionallyselected from the papillary region and the reticular region), subcutis,and conjunctiva) and/or for administration to the eye (e.g., to one ormore of the cornea, sclera, iris, lens, corneal limbus, optic nerve,choroid, ciliary body, anterior segment, anterior chamber, and retina).

In various embodiments, the nucleic acid drug is formulated with one ormore lipids to enhance uptake of the nucleic acid drug by cells, thelipids optionally selected from Table 1. In other embodiments, thenucleic acid drug is formulated with one or more nanoparticles,optionally lipid or polymeric nanoparticles, to enhance uptake of thenucleic acid drug by cells, to enhance duration of protein expression,or to otherwise enhance the safety and/or efficacy of the nucleic aciddrug.

In various embodiments, the nucleic acid drug is administered locally,optionally by one or more of subcutaneous injection, intradermalinjection, subdermal injection and intramuscular injection, and theeffective dose is administered to a surface area of about 4 mm² to about1000 mm² (e.g. about, or no more than about, 4 mm², or 5 mm², or 10 mm²,or 25 mm², or 50 mm², or 75 mm², or 100 mm², or 125 mm², or 150 mm², or200 mm², or 500 mm², or 1000 mm²).

In various embodiments, the nucleic acid drug is administered in atreatment regimen, optionally with an additional agent or adjuvanttherapy described herein, and the administration is about weekly toabout once every 24 weeks (e.g. about, or not more than about, weekly,or once every 2 weeks, or once every 3 weeks, or once every 4 weeks, oronce every 5 weeks, or once every 6 weeks, or once every 7 weeks, oronce every 8 weeks, or once every 9 weeks, or once every 9 weeks, oronce every 9 weeks, or once every 9 weeks, or once every 10 weeks, oronce every 11 weeks, or once every 12 weeks, or once every 13 weeks, oronce every 14 weeks, or once every 15 weeks, or once every 20 weeks, oronce every 24 weeks). In other embodiments, the nucleic acid drug isadministered in a treatment regimen, optionally with an additional agentor adjuvant therapy described herein, and the administration is aboutdaily to about weekly (e.g., about, or not more than about, daily, oronce every 2 days, or once every 3 days, or once every 4 days, or onceevery 5 days, or once every 6 days, or weekly).

In various embodiments the nucleic acid drug comprises RNA comprisingone or more non-canonical nucleotides, optionally having one or moresubstitutions at the 2C and/or 4C and/or 5C positions for a pyrimidineor the 6C and/or 7N and/or 8C positions for a purine. In variousembodiments, the non-canonical nucleotide is one or more of thenon-canonical nucleotides described herein, including, for example,5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine,5-hydroxyuridine, 5-hydroxypseudouridine, 5-methyluridine,5-methylpseudouridine, 5-hydroxymethyluridine,5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine,5-formyluridine, 5-formylpseudouridine, 5-methoxyuridine, and5-methoxypseudouridine. Further, the RNA comprising one or morenon-canonical nucleotides may have one or more of a 5′-UTR comprising aKozak consensus sequence, a 5′-UTR or 3′-UTR comprising a sequence thatincreases RNA stability in vivo (e.g. an alpha-globin or beta-globin5′-UTR or an alpha-globin or beta-globin 3′-UTR), and a 3′ poly(A) tailfrom about 20 nucleotides to about 250 nucleotides in length (e.g. about20, or about 30, or about 40, or about 50, or about 60, or about 70, orabout 80, or about 90, or about 100, or about 110, or about 120, orabout 130, or about 140, or about 150, or about 160, or about 170, orabout 180, or about 190, or about 200, or about 210, or about 220, orabout 230, or about 240, or about 250 nucleotides in length).

Further, some aspects of the methods described herein find use invarious medical treatments, including, by way of illustration, treatinga disease, disorder and/or condition of the integumentary system oraltering, modifying and/or changing the integumentary system (e.g.cosmetically).

Also contemplated are kits suitable for use in human therapy, comprisingthe nucleic acid drug described herein in a unit dosage form of about 10ng to about 5000 ng (e.g. about, or no more than about, 10 ng, or 20 ng,or 50 ng, or 100 ng, or 200 ng, or 300 ng, or 400 ng, or 500 ng, or 600ng, or 700 ng, or 800 ng, or 900 ng, or 1000 ng, or 1100 ng, or 1200 ng,or 1300 ng, or 1400 ng, or 1500 ng, or 1600 ng, or 1700 ng, or 1800 ng,or 1900 ng, or 2000 ng, or 3000 ng, or 4000 ng, or 5000 ng) and aninjection needle.

Further, in some aspects, the present invention provides apharmaceutical formulation comprising the nucleic acid drug describedherein and one or more of the vehicles (a/k/a “transfection reagents”,e.g., lipids) described herein, the formulation optionally beingsuitable for one or more of subcutaneous injection, intradermalinjection, subdermal injection, intramuscular injection, intraocularinjection, intravitreal injection, intra-articular injection,intracardiac injection, intravenous injection, epidural injection,intrathecal injection, intraportal injection, intratumoral injection,and topical administration. As used herein, the term “injection” canrefer to injection, for example, with a syringe, and to other methods ofadministering liquids, for example, infusion, perfusion, administrationusing a pen injector, cartridge system needle-array or patch and/oradministration by a catheter system.

In some aspects, nucleic acid delivery patches are provided. In oneaspect, devices for delivering nucleic acids using electric fields areprovided. Other aspects pertain to methods and compositions for deliveryof nucleic acids to the skin. Still further aspects pertain to methodsand compositions for expression of proteins in the skin.

In one aspect, the invention provides methods and compositions fortreating diseases and conditions in humans, including, but not limitedto, prophylactic treatments, treatments for rare diseases, including,but not limited to, dermatologic rare diseases, and treatments for usein medical dermatology and aesthetic medicine. In another aspect, theinvention provides cosmetics comprising nucleic acids. Still furtheraspects relate to methods and compositions for altering pigmentation,for example, for the treatment of pigmentation disorders. Still furtheraspects relate to methods and compositions for enhancing healing,including, but not limited to, healing in response to a wound orsurgery. The compositions of the present invention may alter, modifyand/or change the appearance of a member of the integumentary system ofa subject such as, but not limited, to skin, hair and nails. Suchalteration, modification and/or change may be in the context oftreatment methods and/or therapeutic uses as described herein including,by way of non-limiting example, dermatological treatments and cosmeticsprocedures.

Further, in various embodiments, the present invention relates to thetargeting of various therapeutic proteins that are not limited todermatological applications. For example, in various embodiments, thepresent compositions and methods find use in methods of treatment thatare mediated by the increased expression of, for example, varioussoluble proteins as illustrated herein. In various embodiments, thenucleic acid drug encodes and/or increases the expression of one or moreof a circulating protein, an extracellular matrix protein, an engineeredprotein, a gene-editing protein, a protein or peptide hormone, anenzyme, erythropoietin, darbepoetin alfa, NOVEPOETIN, elastin, collagen,an antibody or antibody fragment (e.g., a neutralizing antibody orantibody fragment), an intracellular protein, telomerase reversetranscriptase, a membrane protein, a fusion protein, a receptor, aligand binding domain, a protein inhibitor, or a biologically activefragment, analogue or variant thereof. In other embodiments,administration of the nucleic acid drug results in one or more of anincrease in hematocrit, an increase in tissue elasticity, an increase intissue strength, and increase in skin hydration and/or water retention,hair growth, fat reduction, an insertion, deletion or mutation in DNA,conversion of a prodrug to an active drug, a decrease in tumor sizeand/or number, a decrease in plaque size and/or number, an increase invascularization, a decrease in vascularization, an increase in visualacuity, a decrease in pain, an increase in cardiac output (e.g.,ejection fraction and stroke volume), a decrease in abnormal heartrhythm, a decrease in fibrosis, a decrease in one or more adverseneurological symptoms, conditions, or disorders (e.g., depression,dysregulation of appetite, polyphagia, anorexia, dementia, headache,fatigue, numbness, tremors, and dizziness), a decrease in erectiledysfunction, an increase in vitality, an increase in pulmonary function,an increase in kidney function, an increase in liver function, anincrease in insulin sensitivity, a decrease in insulin sensitivity, adecrease in inflammation, an increase in tear production, an improvementin hearing, an increase in auditory perception, a decrease in tinnitus,a reduction in perspiration, partial or total clearance of an infection,an increase in fertility, a decrease in fertility, inhibition orneutralization of a protein, recruitment or stimulation of one or morecomponents of the immune system, lengthening of telomeres, inhibition ofcellular senescence, an increase in replicative potential,reprogramming, proliferation, differentiation, and an increase indifferentiation potential.

In some aspects, RNA molecules with low toxicity and high translationefficiency are provided. In one aspect, a cell-culture medium forhigh-efficiency in vivo transfection, reprogramming, and gene editing ofcells is provided. Other aspects pertain to methods for producing RNAmolecules encoding reprogramming proteins. Still further aspects pertainto methods for producing RNA molecules encoding gene-editing proteins.

In one aspect, the invention provides high-efficiency gene-editingproteins comprising engineered nuclease cleavage domains. In anotheraspect, the invention provides high-fidelity gene-editing proteinscomprising engineered nuclease cleavage domains. Other aspects relate tohigh-efficiency gene-editing proteins comprising engineered DNA-bindingdomains. Still further aspects pertain to high-fidelity gene-editingproteins comprising engineered DNA-binding domains. Still furtheraspects relate to gene-editing proteins comprising engineered repeatsequences. Still further aspects relate to gene-editing proteinscomprising DNA-modification domains. Some aspects relate to methods foraltering the DNA sequence of a cell by transfecting the cell with orinducing the cell to express a gene-editing protein. Other aspectsrelate to methods for altering the DNA sequence of a cell that ispresent in an in vitro culture. Still further aspects relate to methodsfor altering the DNA sequence of a cell that is present in vivo.

In some aspects, the invention provides methods for treating cancercomprising administering to a patient a therapeutically effective amountof a gene-editing protein or a nucleic-acid encoding a gene-editingprotein. In one aspect, the gene-editing protein is capable of alteringthe DNA sequence of a cancer associated gene. In another aspect, thecancer-associated gene is the BIRC5 gene. In other aspects, thegene-editing protein is capable of altering the DNA sequence of a cellto cause the cell to express an antigen or antigen receptor. In oneaspect, the antigen is a tumor antigen. In another aspect, the antigenreceptor binds to a tumor antigen. In yet another aspect, the antigenreceptor is a chimeric antigen receptor. Still other aspects relate totherapeutics comprising nucleic acids and/or cells and methods of usingtherapeutics comprising nucleic acids and/or cells for the treatment of,for example, type 1 diabetes, heart disease, including ischemic anddilated cardiomyopathy, macular degeneration, Parkinson's disease,cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severecombined immunodeficiency, hereditary sensory neuropathy, xerodermapigmentosum, Huntington's disease, muscular dystrophy, amyotrophiclateral sclerosis, Alzheimer's disease, cancer, and infectious diseasesincluding hepatitis and HIV/AIDS. In some aspects, the nucleic acidscomprise RNA. In other aspects, the RNA comprises one or morenon-canonical nucleotides. In still other aspects, the nucleic acids aredelivered to cells using a virus. In some aspects, the virus is areplication-competent virus. In other aspects, the virus is areplication-incompetent virus.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

Any aspect or embodiment disclosed herein can be combined with any otheraspect or embodiment as disclosed herein.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 depicts primary adult human dermal fibroblasts transfected withRNA encoding green fluorescent protein (“GFP”) and comprising theindicated nucleotides.

FIG. 2 depicts the result of a gene-expression analysis of the primaryadult human dermal fibroblasts of FIG. 1 using a one-step real-timeRT-PCR and primers designed to amplify human interferon beta mRNA. Datawere normalized to the untransfected sample (“Neg.”). GAPDH was used asa loading control.

FIG. 3 depicts the results of a gene-expression analysis of cellstransfected with RNA comprising the indicated nucleotides, conducted asin FIG. 2. Data were normalized to the untransfected sample (“Neg.”).GAPDH was used as a loading control.

FIG. 4 depicts the results of a gene-expression analysis of cellstransfected with RNA comprising the indicated nucleotides, conducted asin FIG. 2, and using primers designed to amplify the indicatedtranscripts. Data were normalized to the untransfected sample (“Neg.”).GAPDH was used as a loading control.

FIG. 5 depicts the results of a gene-expression analysis of primaryhuman epidermal keratinocytes transfected with RNA encoding NOVEPOETIN,and comprising the indicated nucleotides, conducted as in FIG. 2. Datawere normalized to the untransfected sample (“Neg.”). GAPDH was used asa loading control.

FIG. 6 depicts intradermal injection of a solution comprising RNAencoding GFP into the ventral forearm of a healthy, 33 year-old, 70 kg,male human subject.

FIG. 7 depicts a region of the ventral forearm of the subject shown inFIG. 6 after treatment with RNA comprising 5-methoxyuridine and encodingGFP (injection sites 1-3) or COL7 (injection site 4). The image wastaken immediately following the final injection.

FIG. 8 depicts the region of FIG. 7, 24 hours after injection.

FIG. 9 depicts the results of fluorescent imaging of the region of FIG.7, using the indicated fluorescent channels. The dose at each injectionsite is also indicated. Images were taken 24 hours after injection.

FIG. 10 depicts the results of fluorescent imaging of the region of FIG.7, using the FITC fluorescent channel. The dose at each injection siteis indicated. Images were taken 48 hours after injection.

FIG. 11 depicts the results of quantitative fluorescent imaging of theregion of FIG. 7, using the FITC fluorescent channel. The horizontalaxis indicates time after injection.

FIG. 12 depicts the results of fluorescent imaging of an independentexperiment in which a region of the ventral forearm of as the subjectshown in FIG. 6 treated with RNA comprising 5-methoxyuridine andencoding GFP. The image was taken 24 hours after injection.

FIG. 13 depicts the results of an ELISA designed to detect darbepoetinalfa in culture media of primary human epidermal keratinocytestransfected with RNA comprising the indicated nucleotides and encodingNOVEPOETIN.

FIG. 14 depicts the results of an ELISA designed to detect darbepoetinalfa in culture media of primary human epidermal keratinocytestransfected with RNA comprising the indicated nucleotides and encodingNOVEPOETIN.

FIG. 15 depicts the results of an ELISA designed to detect darbepoetinalfa in culture media of primary human epidermal keratinocytestransfected with RNA comprising the indicated nucleotides and encodingNOVEPOETIN.

FIG. 16 depicts primary human dermal fibroblasts transfected with RNAcomprising 5-methoxyuridine and encoding hTERT. Cells were fixed andstained using an antibody targeting hTERT 24 hours after transfection.

FIG. 17 depicts primary adult human dermal fibroblasts transfected withRNA encoding green fluorescent protein (“GFP”), prepared and stored asindicated.

FIG. 18 depicts single administration of NOVECRIT induced a rapidincrease and sustained level of NOVEPOIETIN in serum. The Y axis showsconcentration of NOVEPOIETIN protein (mU/mL).

FIG. 19 depicts a single administration of NOVECRIT stimulatederythropoiesis, yielding elevated hematocrit for at least 14 days. Theleft panel shows % hematocrit on the Y axis, while the right panel shows% reticulocytes.

FIG. 20 depicts a table summarizing TNFα, IL-6, and IFNα cytokine levelsin plasma samples collected from a maximum tolerated dose of NOVECRIT inmale Sprague Dawley rats study of Example 35.

FIG. 21 depicts a SURVEYOR assay using the DNA of primary adult humandermal fibroblasts transfected with RNA TALENs targeting the sequenceTGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 467) and TGGCTGTACAGCTACACCCC (SEQ IDNO: 468), located within the COL7A1 gene. The bands present in the +RNAlane indicate editing of a region of the gene that is frequentlyinvolved in dystrophic epidermolysis bullosa.

FIG. 22 depicts a SURVEYOR assay using the DNA of primary adult humandermal fibroblasts transfected with RNA TALENs targeting the sequenceTTCCACTCCTGCAGGGCCCC (SEQ ID NO: 469) and TCGCCCTTCAGCCCGCGTTC (SEQ IDNO:470), located within the COL7A1 gene. The bands present in the +RNAlane indicate editing of a region of the gene that is frequentlyinvolved in dystrophic epidermolysis bullosa.

FIG. 23 shows the immunogenicity of various synthetic RNA constructs inthe context of a gene-editing (i.e. unmodified nucleotides “A,G,U,C”;pseudouridine only “psU”; 5-methylcytidine only “5mC”; bothpseudouridine and 5-methylcytidine “psU+5mC”; and a negative control“neg”).

FIG. 24 shows the gene-editing activity in cells transfected withvarious synthetic RNA constructs (i.e. unmodified nucleotides “A,G,U,C”;pseudouridine only “psU”; 5-methylcytidine only “5mC”; bothpseudouridine and 5-methylcytidine “psU+5mC”; and a negative control“neg”).

FIG. 25 depicts gene editing of the COL7A1 gene in primary humanepidermal keratinocytes transfected with RNA encoding TALENs and asingle-stranded DNA repair template (“RT”) of the indicated length. Thepresence of bands at the locations shown by asterisks (“*”) indicatessuccessful gene editing.

FIG. 26 depicts gene editing of the COL7A1 gene in primary humanepidermal keratinocytes transfected with RNA encoding TALENs and an 80nt single-stranded DNA repair template (“RT”) at the indicated ratios ofRNA to repair template. The presence of bands at the locations shown byasterisks (“*”) indicates successful gene editing.

FIG. 27 depicts gene correction of the COL7A1 gene in primary humanepidermal keratinocytes transfected with RNA encoding TALENs and asingle-stranded DNA repair template (“RT”) of the indicated length. Thepresence of bands at the locations shown by asterisks (“*”) indicatessuccessful gene correction.

FIG. 28 depicts gene correction of the COL7A1 gene in primary humanepidermal keratinocytes transfected with RNA encoding TALENs and an 80nt single-stranded DNA repair template (“RT”) at the indicated ratios ofRNA to repair template. The presence of bands at the locations shown byasterisks (“*”) indicates successful gene correction.

FIG. 29 depicts gene editing (“T7E1”) and correction (“Digestion”) ofthe COL7A1 gene in primary human epidermal keratinocytes transfectedwith RNA encoding TALENs and an 80 nt single-stranded DNA repairtemplate (“RT”). The presence of bands at the locations shown byasterisks (“*”) indicates successful gene editing (“T7E1”) andcorrection (“Digestion”).

FIG. 30 depicts the amount of BMP7 protein secreted by primary humandermal fibroblasts and primary human epidermal keratinocytes transfectedwith RNA encoding the indicated BMP7 variant, measured by ELISA.Asterisks (“*”) indicate saturation of the ELISA assay.

FIG. 31 depicts the amount of parathyroid hormone secreted by primaryhuman epidermal keratinocytes transfected with RNA encoding PTH,measured by ELISA.

FIG. 32 demonstrates that repeated administration of NOVECRIT stimulatederythropoiesis, yielding elevated hematocrit for at least 14 days. Foreach data set, the order of histograms from left to right is: Group 1,Group 2, Group 3, Group 4, and Group 5.

FIG. 33 provides an illustrative experimental design for studying theeffects of RNAs encoding BMP7 variants for the prevention and treatmentof diabetic nephropathy.

FIG. 34 depicts BMP7 protein levels in rats treated with RNAs encodingBMP7 variants as described in Example 42. Error bars indicate SEM (n=6).

FIG. 35 depicts the urine volume, urine albumin, and urine creatininelevels in rats treated with RNAs encoding BMP7 variants as described inExample 42. For each data set, the order of histograms from left toright is: urine volume, urine creatinine, and urine albumin.

FIG. 36 depicts the effect of RNAs encoding BMP7 variants in treatingdiabetic nephropathy as described in Example 42. For each data set, theorder of histograms from left to right is: Group 6—vehicle and Group7—FTB-2 (BMP7 variant A).

FIG. 37, panels A-I, depict the results of gene-expression analysis ofhuman epidermal keratinocytes transfected with RNA encoding BDNF, BMP-2,BMP-6, IL-2, IL-6, IL-15, IL-22, LIF or FGF-21.

FIG. 38 depicts the results of gene-expression analysis of humanepidermal keratinocytes transfected with RNA encoding IL-15 or IL-15 andIL-15RA.

FIG. 39 depicts the serum levels of FGF21, IL15, IL6, IL22, andNovepoietin following a single intradermal injection of various RNAsencoding these proteins as described in Example 45. Three rats wereanalyzed for each RNA tested.

FIG. 40 depicts the results of the wound-healing assay described inExample 46. Four scratch locations were measured for each well, and twowells were measured for each condition. Error bars indicate standarddeviation.

FIG. 41 depicts gene editing of primary human epidermal keratinocytes,as described in Example 47.

FIG. 42 depicts gene correction of primary human epidermalkeratinocytes, as described in Example 47.

FIG. 43 depicts gene editing of primary human epidermal keratinocytes,as described in Example 47.

FIG. 44 depicts IL10 production in COLO 205 human colorectaladenocarcinoma cells. COLO 205 cells were plated at a density of 20,000cells/well in a 24 well plate in 0.5 mL RPMI1640+10% FBS per well. Thenext day, cells were either (i) transfected with 0.4 μg RNA encodingGFP, as described in example (Example 3), (ii) transfected with 0.4 μgRNA encoding IL22, as described in example (Example 3), (iii) exposed tocell culture medium taken from human epidermal keratinocytes transfectedwith RNA encoding IL22, (iv) exposed to cell culture medium taken fromuntransfected human epidermal keratinocytes, (v) exposed to recombinanthuman IL22 (782-IL-010; R&D Systems). 48 h later, IL10 levels in theculture medium were measured via ELISA (D1000B; R&D Systems). IL22levels for sample (iii) were determined via ELISA (D2200; R&D Systems)prior to the experiment, and the same volumes of medium were used forcondition (iv).

FIG. 45 depicts a BMP7 activity assay involving the measurement ofalkaline phosphatase activity in ATDC 5 cells following transfectionwith RNA encoding BMP7 (variant A) or exposure to recombinant humanBMP7. ATDC 5 cells were plated at a density of 20,000 cells/well in a 24well plate in DMEM/F12+5% FBS. The next day, the cells were serumstarved by changing the medium to DMEM/F12+2% FBS. The following day,the cells were either (i) transfected with RNA encoding BMP7 or (ii)exposed to recombinant human BMP7 (54-BP-010, R&D Systems). Three dayslater, intracellular alkaline phosphatase activity was measured(ab83369; Abcam).

FIG. 46 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) weretransfected with 2 μg RNA encoding the indicated gene-editing proteins(1 μg L and 1 μg R). DNA was harvested after 48 h and analyzed for geneediting (A/B indicates RIBOSLICE_A L, RIBOSLICE_B R; RIBOSLICE Aindicates repeat sequences comprising the sequence: GHGG, HG, GHGG, HG,etc.; RIBOSLICE B indicates repeat sequences comprising the sequence:HG, GHGG, HG, GHGG, etc.; L targets the sequence: TGCCTGGTCCCTGTCTCCCT(SEQ ID NO: 615); R targets the sequence: TGTCTTCTGGGCAGCATCTC (SEQ IDNO: 616); a target sequence is approximately 75 bp from A1AT [SERPINA1]start codon). “TAL” indicates a control TALEN directed to the targetsequence.

FIG. 47 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) weretransfected with 2 μg RNA encoding the indicated gene-editing proteins(1 μg L and 1 μg R). DNA was harvested after 48 h and analyzed for geneediting (A/B indicates RIBOSLICE_A L, RIBOSLICE_B R; RIBOSLICE Aindicates repeat sequences comprising the sequence: GHGG, HG, GHGG, HG,etc.; RIBOSLICE B indicates repeat sequences comprising the sequence:HG, GHGG, HG, GHGG, etc.; L targets the sequence: TATTCCCGGGCTCCCAGGCA(SEQ ID NO: 622); R targets the sequence: TCTCCTGGCCTTCCTGCCTC (SEQ IDNO: 612); a target sequence is near the end of exon 73 of COL7A1). “TAL”indicates a control TALEN directed to the target sequence.

FIG. 48 depicts the results of an experiment in which 50,000 primaryhuman neonatal epidermal keratinocytes (HEKn) (animal-protein free) weretransfected with 2 μg RNA encoding the indicated gene-editing proteins.DNA was harvested after 48 h and analyzed for gene editing (“Neg”indicates untreated HEKn DNA; “WT” indicates wild type FokI; “EA”indicates enhanced activity FokI (S35P and K58E); “Het” indicatesheterodimer (L: Q103E, N113D, I116L, R: E107K, H154R, 1155K); “EA/Het”indicates both EA and Het; L targets the sequence: TATTCCCGGGCTCCCAGGCA(SEQ ID NO: 622); R targets the sequence: TCTCCTGGCCTTCCTGCCTC (SEQ IDNO: 612); a target sequence is near the end of exon 73 of COL7A1).

FIG. 49 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) per well ofa 6-well plate were transfected with 2 ug RNA encoding hGDF15. Theculture medium was analyzed for GDF15 at the indicated time aftertransfection by ELISA (R&D DGD150).

FIG. 50 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) per well ofa 6-well plate were transfected with 2 ug RNA encoding the indicatedprotein. The culture medium was analyzed for IFKB at the indicated timeafter transfection by ELISA (Abcam ab176644). For each time point, thehistograms indicate (left to right): IFKB WT, IFKB S32A, S36A, and Neg.

FIG. 51 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) per well ofa 6-well plate were transfected with 2 μg RNA encoding the indicatedprotein. Images were taken 24 h after transfection.

FIG. 52 depicts the results of an experiment in which 20,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) per well ofa 24-well plate were transfected with 0.2 μg RNA encoding SOD3. Cellswere fixed and stained 24 h after transfection with a 1:100 dilution ofNBP2-38493 (Novus) rabbit anti human SOD3 primary antibody and a 1:1000dilution of 488 donkey anti-rabbit secondary antibody. “BF” indicatesbrightfield and “FL” indicates fluorescence.

FIG. 53 depicts the results of an experiment in which ZDF rats wereadministered RNA encoding hGDF15 by intradermal injection on Days 1, 8,and 15 (Group 4, grey curve (bottom)) or with vehicle only (Group 3,black curve (top)). ALT serum levels were measured at the indicatedtimes.

FIG. 54 depicts the results of an experiment in which ZDF rats wereadministered RNA encoding hGDF15 by intradermal injection on Days 1, 8,and 15 (Group 4, grey curve (bottom)) or with vehicle only (Group 3,black curve (top)). AST serum levels were measured at the indicatedtimes.

FIG. 55 depicts the results of an experiment in which ZDF rats wereadministered RNA encoding hGDF15 by intradermal injection on Days 1, 8,and 15 (Group 4, grey curve (top)) or with vehicle only (Group 3, blackcurve (bottom)). Total cholesterol serum levels were measured at theindicated times.

FIG. 56 depicts the results of an experiment in which ZDF rats wereadministered RNA encoding hGDF15 by intradermal injection on Days 1, 8,and 15 (Group 4, grey (bottom at day 7)) or with vehicle only (Group 3,black (top at day 7)). Glucose serum levels were measured at theindicated times.

FIG. 57 depicts the results of an experiment in which ZDF rats wereadministered RNA encoding hGDF15 by intradermal injection on Days 1, 8,and 15 (Group 4, grey (top)) or with vehicle only (Group 3, black(bottom)). Triglycerides serum levels were measured at the indicatedtimes.

FIG. 58 depicts the results of an experiment in which ZDF rats wereadministered RNA encoding hGDF15 by intradermal injection on Days 1, 8,and 15 (treatment group) or with vehicle only (control group). % changeof ALT, AST, Total cholesterol, Glucose, and Triglycerides serum levelsin treatment group vs control group are shown.

FIG. 59 depicts the results of an experiment in which leanSprague-Dawley rats were administered RNA encoding hGDF15 by intradermalinjection on Days 1, 8, and 15 (Group 2) or with vehicle only (Group 1)and ZDF rats were administered RNA encoding hGDF15 by intradermalinjection on Days 1, 8, and 15 (Group 4) or with vehicle only (Group 3).GDF15 serum levels were measured at the indicated times. For eachtimepoint, the histograms indicate (left to right) Group 1, Group 2,Group 3, and Group 4.

FIG. 60 depicts the results of an experiment in which 200,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) per well ofa 6-well plate were transfected with 2 μg RNA encoding hESM1. Theculture medium was analyzed for ESM1 52 hours after transfection byELISA (Abcam ab213776).

FIG. 61 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) weretransfected with 2 μg RNA encoding the HBB exon 1 TALEN L and HBB exon 1TALEN R gene-editing proteins (1 μg each). DNA was harvested after 48 hand analyzed for gene editing (T7E1 assay; forward primer:gccaaggacaggtacggctgtcatc (SEQ ID NO: 627); reverse primer:cttgccatgagccttcaccttagggttg (SEQ ID NO: 628); product size: 518nt;predicted band sizes: 300nt, 218nt).

FIG. 62 depicts the results of an experiment in which 100,000 primaryhuman neonatal epidermal keratinocytes (animal-protein free) weretransfected with 2 μg RNA encoding the PD1 exon 1 TALEN L and PD1 exon 1TALEN R gene-editing proteins (1 μg each). DNA was harvested after 48 hand analyzed for gene editing (T7E1 assay; forward primer:tcctctgtctccctgtctctgtctctctctc (SEQ ID NO: 594); reverse primer:ggacttgggccaggggaggag (SEQ ID NO: 595); product size: 612nt; predictedband sizes: 349nt, 263nt).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of a safe andeffective dosing strategy for nucleic acid drugs, including RNA, such asRNA comprising non-canonical (or “modified”) nucleotides, in humans. Theinventors believe this to be the first report of safe and effectivedosing of RNA molecules, including those comprising non-canonicalnucleotides, in humans. Despite reports in the art that very large dosesof RNA molecules are needed for mammalian dosing, and minimaltherapeutic effect is achieved despite high dosing (see, e.g. US PatentPublication No. 2013/0245103), the present inventors have surprisinglymanaged to dose synthetic RNA in a human and achieve significant targetprotein expression with minimal immunological or other side effects.

In various embodiments, the present invention provides improved doses,formulations, administration and methods of use of nucleic acid drugs,which include RNA, which may contain non-canonical nucleotides (e.g. aresidue other than adenine, guanine, thymine, uracil, and cytosine orthe standard nucleoside, nucleotide, deoxynucleoside or deoxynucleotidederivatives thereof). In various embodiments, the RNA comprisingnon-canonical nucleotides leads to the expression of a protein encodedby the RNA, the protein often being one of therapeutic benefit(sometimes called the “target” or “protein of interest”). Further, thisexpression of therapeutic protein is achieved with minimal or negligibletoxicity.

In various aspects, the present invention is based on the surprisingdiscovery of safe and effective doses and administration parameters ofnucleic acid drugs for human subjects. Nucleic acid drugs include adsDNA molecule, a ssDNA molecule, a RNA molecule, a dsRNA molecule, assRNA molecule, a plasmid, an oligonucleotide, a synthetic RNA molecule,a miRNA molecule, an mRNA molecule, and an siRNA molecule. In variousembodiments, the RNA comprises non-canonical nucleotides.

In some aspects, there is provided a method for delivering a nucleicacid drug, comprising administering an effective dose of a nucleic aciddrug to a human subject in need thereof, wherein the nucleic acid drugcomprises a synthetic RNA. In various embodiments, the effective dose isan amount sufficient to substantially increase an amount of a proteinencoded by the nucleic acid drug in the human subject. For example, whenthe nucleic acid drug is a synthetic RNA comprising one or more modifiednucleotides, the nucleic acid drug may result in higher proteinexpression than levels obtainable with a nucleic acid drug that does notcomprise one or more modified nucleotides (e.g., RNA comprising thecanonical nucleotides A, G, U, and C). In some embodiments, the nucleicacid drug results in about a 2-fold, or about a 3-fold, or about a4-fold, or about a 5-fold, or about a 10-fold, or about a 15-fold, orabout a 20-fold, or about a 25-fold, or about a 30-fold, or about a35-fold, or about a 40-fold, or about a 45-fold, or about a 50-fold, orabout a 100-fold increase in protein expression as compared to levelsobtainable with a nucleic acid drug that does not comprise one or moremodified nucleotides.

In some embodiments, the nucleic acid drug provides a sustainedtherapeutic effect that is optionally mediated by a sustained expressionof target protein. For instance, in some embodiments, the therapeuticeffect is present for over about 1 day, or over about 2 days, or overabout 3 days, or over about 4 days, or over about 5 days, or over about6 days, or over about 7 days, or over about 8 days, or over about 9days, or over about 10 days, or over about 14 days after administration.In some embodiments, this sustained effect obviates the need for, orreduces the amount of, maintenance doses.

In some embodiments, the nucleic acid drug provides a sustained targetprotein level. For instance, in some embodiments, the target protein ispresent (e.g. in measurable amounts, e.g. in the serum of a patient towhom the nucleic acid drug has been administered) for over about 1 day,or over about 2 days, or over about 3 days, or over about 4 days, orover about 5 days, or over about 6 days, or over about 7 days, or overabout 8 days, or over about 9 days, or over about 10 days, or over about14 days after administration. In some embodiments, this sustained effectobviates the need for, or reduces the amount of, maintenance doses.

In various embodiments, the nucleic acid drug provides therapeuticaction without sustained presence of the nucleic acid drug itself. Insome embodiments, the nucleic acid drug is rapidly metabolized, forinstance, within about 6 hours, or about 12 hours, or about 18 hours, orabout 24 hours, or about 2 days, or about 3 days, or about 4 days, orabout 5 days, or about 1 week from administration.

In various embodiments, the effective dose is an amount thatsubstantially avoids cell toxicity in vivo. In various embodiments, theeffective dose is an amount that substantially avoids an immune reactionin a human subject. For example, the immune reaction may be an immuneresponse mediated by the innate immune system. Immune response can bemonitored using markers known in the art (e.g. cytokines, interferons,TLRs). In some embodiments, the effective dose obviates the need fortreatment of the human subject with immune suppressants agents (e.g.B18R) used to moderate the residual toxicity. Accordingly, in someembodiments, the present methods allow for dosing that providesincreased protein expression and reduces toxicity.

In some embodiments, the immune response is reduced by about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, about 99%, about 99.9%, or greater than about99.9% as compared to the immune response induced by a correspondingunmodified nucleic acid. In some embodiments, upregulation of one ormore immune response markers is reduced by about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, about 99%, about 99.9%, or greater than about 99.9% ascompared to the upregulation of the one or more immune response markersinduced by a corresponding unmodified nucleic acid. In some embodiments,the immune response marker comprises an mRNA or protein product of aninterferon gene, including an interferon alpha gene, IFNB1, TLR3,RARRES3, EIF2AK2, STAT1, STAT2, IFIT1, IFIT2, IFIT3, IFIT5, OAS1, OAS2,OAS3, OASL, ISG20 or a fragment, variant, analogue, or family-memberthereof. In some embodiments, the immune response marker comprises anmRNA or protein product of an TNF gene, including an TNF alpha gene,TNFRSF1A; TNFRSF1B; LTBR; TNFRSF4; CD40; FAS; TNFRSF6B; CD27; TNFRSF8;TNFRSF9; TNFRSF10A; TNFRSF10B; TNFRSF10C; TNFRSF10D; TNFRSF11A;TNFRSF11B; TNFRSF12A; TNFRSF13B; TNFRSF13C; TNFRSF14; NGFR; TNFRSF17;TNFRSF18; TNFRSF19; TNFRSF21; TNFRSF25; and EDA2R or a fragment,variant, analogue, or family-member thereof. In some embodiments, theimmune response marker comprises an mRNA or protein product of aninterleukin gene, including an IL-6 gene, IL-1; IL-2; IL-3; IL-4; IL-5;IL-6; IL-7; IL-8 or CXCL8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14;IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24;IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35;IL-36 or a fragment, variant, analogue, or family-member thereof.

In some embodiments, cell death is about 10%, about 25%, about 50%,about 75%, about 85%, about 90%, about 95%, or over about 95% less thanthe cell death observed with a corresponding unmodified nucleic acid.Moreover, cell death may affect fewer than about 50%, about 40%, about30%, about 20%, about 10%, about 5%, about 1%, about 0.1%, about 0.01%or fewer than about 0.01% of cells contacted with the modified nucleicacids.

In some embodiments, there is provided a method for expressing a proteinof interest in a population of cells in a mammalian subject, comprisingadministering a non-viral transfection composition comprising aneffective dose of a RNA encoding the protein of interest to said cells,the RNA containing one or more non-canonical nucleotides that avoidsubstantial cellular toxicity, where the transfection composition isadministered in an amount that allows for expression of said protein insaid cells for at least about five days (e.g. about 5, or about 6, orabout 7, about 8, or about 9, or about 10, or about 14 days) withoutsubstantial cellular toxicity. In some embodiments, there is provided amethod for expressing a protein of interest in a population of cells ina mammalian subject, comprising administering a non-viral transfectioncomposition comprising an effective dose of a RNA encoding the proteinof interest to said cells, the RNA containing one or more non-canonicalnucleotides that avoid substantial cellular toxicity, where thetransfection composition is administered in an amount that allows forexpression of said protein in said cells for at least about six hours(e.g. about six hours, or about 12 hours, or about 1 day, or about 2days, or about 3 days, or about 4 days, or about 5 days) withoutsubstantial cellular toxicity.

In some embodiments, the effective dose of the nucleic acid drug,including synthetic RNA, is about 100 ng to about 2000 ng, or about 200ng to about 1900 ng, or about 300 ng to about 1800 ng, or about 400 ngto about 1700 ng, or about 500 ng to about 1600 ng, or about 600 ng toabout 1500 ng, or about 700 ng to about 1400 ng, or about 800 ng toabout 1300 ng, or about 900 ng to about 1200 ng, or about 1000 ng toabout 1100 ng, or about 500 ng to about 2000 ng, or about 500 ng toabout 1500 ng, or about 500 ng to about 1000 ng, or about 1000 ng toabout 1500 ng, or about 1000 ng to about 2000 ng, or about 1500 ng toabout 2000 ng, or about 100 ng to about 500 ng, or about 200 ng to about400 ng, or about 10 ng to about 100 ng, or about 20 ng to about 90 ng,or about 30 ng to about 80 ng, or about 40 ng to about 70 ng, or about50 ng to about 60 ng.

In some embodiments, the effective dose of the nucleic acid drug,including synthetic RNA, is no more than about 50 ng, or about 100 ng,or about 200 ng, or about 300 ng, or about 400 ng, or about 500 ng, orabout 600 ng, or about 700 ng, or about 800 ng, or about 900 ng, orabout 1000 ng, or about 1100 ng, or about 1200 ng, or about 1300 ng, orabout 1400 ng, or about 1500 ng, or about 1600 ng, or about 1700 ng, orabout 1800 ng, or about 1900 ng, or about 2000 ng, or about 3000 ng, orabout 4000 ng, or about 5000 ng.

In some embodiments, the effective dose of the nucleic acid drug,including synthetic RNA, is about 50 ng, or about 100 ng, or about 200ng, or about 300 ng, or about 400 ng, or about 500 ng, or about 600 ng,or about 700 ng, or about 800 ng, or about 900 ng, or about 1000 ng, orabout 1100 ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, orabout 1500 ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, orabout 1900 ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, orabout 5000 ng.

In some embodiments, the effective dose of the nucleic acid drug,including synthetic RNA, is about 0.028 pmol, or about 0.05 pmol, orabout 0.1 pmol, or about 0.2 pmol, or about 0.3 pmol, or about 0.4 pmol,or about 0.5 pmol, or about 0.6 pmol, or about 0.7 pmol, or about 0.8pmol, or about 0.9 pmol, or about 1.0 pmol, or about 1.2 pmol, or about1.4 pmol, or about 1.6 pmol, or about 1.8 pmol, or about 2.0 pmol, orabout 2.2 pmol, or about 2.4 pmol, or about 2.6 pmol, or about 2.8 pmol,or about 3.0 pmol, or about 3.2 pmol, or about 3.4 pmol, or about 3.6pmol, or about 3.8 pmol, or about 4.0 pmol, or about 4.2 pmol, or about4.4 pmol, or about 4.6 pmol, or about 4.8 pmol, or about 5.0 pmol, orabout 5.5 pmol, or about 5.7 pmol.

In some embodiments, the nucleic acid drug, including synthetic RNA, isadministered at a concentration of about 0.1 nM, or about 0.25 nM, orabout 0.5 nM, or about 0.75 nM, or about 1 nM, or about 2.5 nM, or about5 nM, or about 7.5 nM, or about 10 nM, or about 20 nM, or about 30 nM,or about 40 nM, or about 50 nM, or about 60 nM, or about 70 nM, or about80 nM, or about 90 nM, or about 100 nM, or about 110 nM, or about 120nM, or about 150 nM, or about 175 nM, or about 200 nM.

In some embodiments, the effective dose of the nucleic acid drug isabout 350 ng/cm², or about 500 ng/cm², or about 750 ng/cm², or about1000 ng/cm², or about 2000 ng/cm², or about 3000 ng/cm², or about 4000ng/cm², or about 5000 ng/cm², or about 6000 ng/cm², or about 7000ng/cm². In other embodiments, the effective dose is less than about 350ng/cm². In certain embodiments, the effective dose is about 35 ng/cm²,or about 50 ng/cm², or about 75 ng/cm², or about 100 ng/cm², or about150 ng/cm², or about 200 ng/cm², or about 250 ng/cm², or about 300ng/cm², or about 350 ng/cm².

In some embodiments, the effective dose of the nucleic acid drug isabout 35 ng/cm² to about 7000 ng/cm², or about 50 ng/cm² to about 5000ng/cm², or about 100 ng/cm² to about 3000 ng/cm², or about 500 ng/cm² toabout 2000 ng/cm², or about 750 ng/cm² to about 1500 ng/cm², or about800 ng/cm² to about 1200 ng/cm², or about 900 ng/cm² to about 1100ng/cm².

In some embodiments, the effective dose of the nucleic acid drug isabout 1 picomole/cm², or about 2 picomoles/cm², or about 3picomoles/cm², or about 4 picomoles/cm², or about 5 picomoles/cm², orabout 6 picomoles/cm², or about 7 picomoles/cm², or about 8picomoles/cm², or about 9 picomoles/cm², or about 10 picomoles/cm², orabout 12 picomoles/cm², or about 14 picomoles/cm², or about 16picomoles/cm², or about 18 picomoles/cm², or about 20 picomoles/cm². Inother embodiments, the effective dose is less than about 1 picomole/cm².In certain embodiments, the effective dose is about 0.1 picomoles/cm²,or about 0.2 picomoles/cm², or about 0.3 picomoles/cm², or about 0.4picomoles/cm², or about 0.5 picomoles/cm², or about 0.6 picomoles/cm²,or about 0.7 picomoles/cm², or about 0.8 picomoles/cm², or about 0.9picomoles/cm², or about 1 picomole/cm².

In some embodiments, the effective dose of the nucleic acid drug isabout 0.1 picomoles/cm² to about 20 picomoles/cm², or about 0.2picomoles/cm² to about 15 picomoles/cm², or about 0.5 picomoles/cm² toabout 10 picomoles/cm², or about 0.8 picomoles/cm² to about 8picomoles/cm², or about 1 picomole/cm² to about 5 picomoles/cm², orabout 2 picomoles/cm² to about 4 picomoles/cm².

In various embodiments, the nucleic acid drug, including synthetic RNA,is administered in a pharmaceutically acceptable formulation. In variousembodiments, the nucleic acid drug, including synthetic RNA, isformulated for one or more of injection and topical administration. Byway of example, the nucleic acid drug, including synthetic RNA, may beformulated for injection to a tissue of interest, e.g. a disease site(by way of non-limiting example, a tumor). In various embodiments,injection includes delivery via a patch. In some embodiments, thedelivery is mediated by electrical stimulation. In various embodiments,the nucleic acid drug, including synthetic RNA, is formulated foradministration to one or more of the epidermis (optionally selected fromthe stratum corneum, stratum lucidum, stratum granulosum, stratumspinosum, and stratum germinativum), basement membrane, dermis(optionally selected from the papillary region and the reticularregion), subcutis, conjunctiva cornea, sclera, iris, lens, corneallimbus, optic nerve, choroid, ciliary body, anterior segment, anteriorchamber, and retina. In various embodiments, the nucleic acid drug,including synthetic RNA, is formulated for one or more of subcutaneousinjection, intradermal injection, subdermal injection, intramuscularinjection, intraocular injection, intravitreal injection,intra-articular injection, intracardiac injection, intravenousinjection, epidural injection, intrathecal injection, intraportalinjection, intratumoral injection, and topical administration. Invarious embodiments, the nucleic acid drug, including synthetic RNA, isformulated for intradermal (ID) injection to one or more of the dermisor epidermis. In various embodiments, the nucleic acid drug, includingsynthetic RNA, is administered in a manner such that it effects one ormore of keratinocytes and fibroblasts (e.g. causes these cells toexpress one or more therapeutic proteins).

Accordingly, the present invention provides various formulations asdescribed herein. Further, in some embodiments, the formulationsdescribed herein find use in the various delivery and/or treatmentmethods of the present invention. For instance, formulations cancomprise a vesicle, for instance, a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989). In various embodiments, the formulation comprises anaqueous suspension of liposomes. Illustrative liposome components areset forth in Table 1, and are given by way of example, and not by way oflimitation. In various embodiments, one or more, or two or more, orthree or more, or four or more, or five or more of the lipids of Table 1are combined in a formulation.

TABLE 1 Illustrative Biocompatible Lipids 13β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol)2 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP/18:1 TAP) 3N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan- 1-aminium(DOBAQ) 4 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) 51,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP) 61,2-stearoyl-3-trimethylammonium-propane (18:0 TAP) 71,2-dioleoyl-3-dimethylammonium-propane (DODAP/18:1 DAP) 81,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP) 91,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP) 101,2-distearoyl-3-dimethylammonium-propane (18:0 DAP) 11dimethyldioctadecylammonium (18:0 DDAB) 121,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EthylPC) 131,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EthylPC) 141,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EthylPC) 151,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EthylPC) 161,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EthylPC) 171,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EthylPC) 181-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:1-18:1EthylPC) 19 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) 20N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5)21 2,3-dioleyloxy-N-[2-spermine carboxamide]ethyl-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA) 221,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER) 23N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2- hydroxyethyl)ammoniumbromide (DMRIE) 24 LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINERNAiMAX, LIPOFECTAMINE 3000, LIPOFECTAMINE MessengerMAX, TransIT mRNA 25dioctadecyl amidoglyceryl spermine (DOGS) 26 dioleoyl phosphatidylethanolamine (DOPE)

In some embodiments, the liposomes include LIPOFECTAMINE 3000. In someembodiments, the liposomes include one or more lipids described in U.S.Pat. Nos. 4,897,355 or 7,479,573 or in International Patent PublicationNo. WO/2015/089487, or in Feigner, P. L. et al. (1987) Proc. Natl. Acad.Sci. USA 84:7413-7417, the entire contents of each is incorporated byreference in their entireties).

In some embodiments, the liposome comprisesN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).In some embodiments, the liposome comprisesdioleoylphosphatidylethanolamine (DOPE).

In one embodiment, the liposomes include one or more polyethylene glycol(PEG) chains, optionally selected from PEG200, PEG300, PEG400, PEG600,PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In someembodiments, the PEG is PEG2000. In some embodiments, the liposomesinclude 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or aderivative thereof.

In some embodiments, the formulation comprises one or more ofN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),fully hydrogenated phosphatidylcholine, cholesterol, LIPOFECTAMINE 3000,a cationic lipid, a polycationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (FA-MPEG5000-DSPE).

In one embodiment, the formulation comprises about 3.2 mg/mLN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),about 9.6 mg/mL fully hydrogenated phosphatidylcholine, about 3.2 mg/mLcholesterol, about 2 mg/mL ammonium sulfate, and histidine as a buffer,with about 0.27 mg/mL1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (FA-MPEG5000-DSPE) added to the lipid mixture. In anotherembodiment, the nucleic acids are complexed by combining 1 μL ofLIPOFECTAMINE 3000 per about 1 μg of nucleic acid and incubating at roomtemperature for at least about 5 minutes. In one embodiment, theLIPOFECTAMINE 3000 is a solution comprising a lipid at a concentrationof about 1 mg/mL. In some embodiments, nucleic acids are encapsulated bycombining about 10 μg of the liposome formulation per about 1 μg ofnucleic acid and incubating at room temperature for about 5 minutes.

In some embodiments, the formulation comprises one or morenanoparticles. In one embodiment, the nanoparticle is a polymericnanoparticle. In various embodiments, the formulation comprises one ormore of a diblock copolymer, a triblock copolymer, a tetrablockcopolymer, and a multiblock copolymer. In various embodiments, theformulation comprises one or more of polymeric nanoparticles comprisinga polyethylene glycol (PEG)-modified polylactic acid (PLA) diblockcopolymer (PLA-PEG) and PEG-polypropylene glycol-PEG-modifiedPLA-tetrablock copolymer (PLA-PEG-PPG-PEG).

In some embodiments, the formulation comprises one or more lipids thatare described in WO/2000/027795, the entire contents of which are herebyincorporated by reference.

In one embodiment, the therapeutic comprises one or more ligands. Inanother embodiment, the therapeutic comprises at least one of: androgen,CD30 (TNFRSF8), a cell-penetrating peptide, CXCR, estrogen, epidermalgrowth factor, EGFR, HER2, folate, insulin, insulin-like growthfactor-I, interleukin-13, integrin, progesterone,stromal-derived-factor-1, thrombin, vitamin D, and transferrin or abiologically active fragment or variant thereof.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal, intraportall orintravenous injection, or by direct injection into diseased, e.g.cancer, tissue. The agents disclosed herein may also be administered bycatheter systems. Such compositions would normally be administered aspharmaceutically acceptable compositions as described herein.

Administration of the compositions described herein may be, for example,by injection, topical administration, ophthalmic administration andintranasal administration. The injection, in some embodiments, may belinked to an electrical force (e.g. electroporation, including withdevices that find use in electrochemotherapy (e.g. CLINIPORATOR, IGEASrI, Carpi [MO], Italy)). The topical administration may be, but is notlimited to, a cream, lotion, ointment, gel, spray, solution and thelike. The topical administration may further include a penetrationenhancer such as, but not limited to, surfactants, fatty acids, bilesalts, chelating agents, non-chelating non-surfactants,polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, fattyacids and/or salts in combination with bile acids and/or salts, sodiumsalt in combination with lauric acid, capric acid and UDCA, and thelike. The topical administration may also include a fragrance, acolorant, a sunscreen, an antibacterial and/or a moisturizer. Thecompositions described herein may be administered to at least one sitesuch as, but not limited to, forehead, scalp, hair follicles, hair,upper eyelids, lower eyelids, eyebrows, eyelashes, infraorbital area,periorbital areas, temple, nose, nose bridge, cheeks, tongue, nasolabialfolds, lips, periobicular areas, jaw line, ears, neck, breast, forearm,upper arm, palm, hand, finger, nails, back, abdomen, sides, buttocks,thigh, calf, feet, toes and the like.

Routes of administration include, for example: intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,intraportal, rectally, by inhalation, or topically, particularly to theears, nose, eyes, or skin. In some embodiments, the administering iseffected orally or by parenteral injection.

Upon formulation, solutions may be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective, as described herein. The formulations may easily beadministered in a variety of dosage forms such as injectable solutions,drug release capsules and the like. For parenteral administration in anaqueous solution, for example, the solution generally is suitablybuffered and the liquid diluent first rendered isotonic with, forexample, sufficient saline or glucose. Such aqueous solutions may beused, for example, for intravenous, intramuscular, subcutaneous andintraperitoneal administration. Preferably, sterile aqueous media areemployed as is known to those of skill in the art, particularly in lightof the present disclosure.

In various embodiments, the nucleic acid drug, including RNA comprisingone or more non-canonical nucleotides, and/or formulations comprisingthe same, is administered locally, optionally by one or more ofsubcutaneous injection, intradermal injection, subdermal injection andintramuscular injection, and the effective dose is administered to asurface area of about 4 mm² to about 150 mm² (e.g. about, or no morethan about, 4 mm², or about 5 mm², or about 6 mm², or about 7 mm², orabout 8 mm², or about 10 mm², or about 20 mm², or about 50 mm², or about100 mm², or about 150 mm²). In various embodiments, the nucleic aciddrug, including RNA comprising one or more non-canonical nucleotides,and/or formulations comprising the same, is administered locally,optionally by one or more of subcutaneous injection, intradermalinjection, subdermal injection and intramuscular injection, and theeffective dose administered to a surface area of no more than about 4mm², or about 5 mm², or about 6 mm², or about 7 mm², or about 8 mm², orabout 10 mm², or about 20 mm², or about 50 mm², or about 100 mm², orabout 150 mm². In various embodiments, the nucleic acid drug, includingRNA comprising one or more non-canonical nucleotides, and/orformulations comprising the same, is administered locally, optionally byone or more of subcutaneous injection, intradermal injection, subdermalinjection and intramuscular injection, and the effective doseadministered to a surface area of about 4 mm², or about 5 mm², or about6 mm², or about 7 mm², or about 8 mm², or about 10 mm², or about 20 mm²,or about 50 mm², or about 100 mm², or about 150 mm².

In various embodiments, the nucleic acid drug, including RNA comprisingone or more non-canonical nucleotides, and/or formulations comprisingthe same, is administered locally, optionally by one or more ofsubcutaneous injection, intradermal injection, subdermal injection andintramuscular injection, and the effective dose (weight RNA/surface areaof injection) is about 35 ng/cm² to about 7000 ng/cm². In variousembodiments, the nucleic acid drug, including RNA comprising one or morenon-canonical nucleotides, and/or formulations comprising the same, isadministered locally, optionally by one or more of subcutaneousinjection, intradermal injection, subdermal injection and intramuscularinjection, and the effective dose (weight RNA/surface area of injection)is no more than about 35 ng/cm², or about 50 ng/cm², or about 75 ng/cm²,or about 100 ng/cm², or about 125 ng/cm², or about 150 ng/cm², or about175 ng/cm², or about 200 ng/cm², or about 225 ng/cm², or about 250ng/cm², or about 500 ng/cm², or about 1000 ng/cm², or about 2000 ng/cm²,or about 5000 ng/cm², or about 7000 ng/cm². In various embodiments, thenucleic acid drug, including RNA comprising one or more non-canonicalnucleotides, and/or formulations comprising the same, is administeredlocally, optionally by one or more of subcutaneous injection,intradermal injection, subdermal injection and intramuscular injection,and the effective dose (weight RNA/surface area of injection) is about35 ng/cm², or about 50 ng/cm², or about 75 ng/cm², or about 100 ng/cm²,or about 125 ng/cm², or about 150 ng/cm², or about 175 ng/cm², or about200 ng/cm², or about 225 ng/cm², or about 250 ng/cm², or about 500ng/cm², or about 1000 ng/cm², or about 2000 ng/cm², or about 5000ng/cm², or about 7000 ng/cm².

Pharmaceutical preparations may additionally comprise delivery reagents(a.k.a. “transfection reagents”, a.k.a. “vehicles”, a.k.a. “deliveryvehicles”) and/or excipients. Pharmaceutically acceptable deliveryreagents, excipients, and methods of preparation and use thereof,including methods for preparing and administering pharmaceuticalpreparations to patients (a.k.a. “subjects”) are well known in the art,and are set forth in numerous publications, including, for example, inUS Patent Appl. Pub. No. US 2008/0213377, the entirety of which isincorporated herein by reference.

For example, the present compositions can be in the form ofpharmaceutically acceptable salts. Such salts include those listed in,for example, J. Pharma. Sci. 66, 2-19 (1977) and The Handbook ofPharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are herebyincorporated by reference in their entirety. Non-limiting examples ofpharmaceutically acceptable salts include: sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate,phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate,α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate,caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate,malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate,phthalate, teraphthalate, propiolate, propionate, phenylpropionate,sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate,ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, tartarate salts, hydroxidesof alkali metals such as sodium, potassium, and lithium; hydroxides ofalkaline earth metal such as calcium and magnesium; hydroxides of othermetals, such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines,dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-0H-loweralkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

The present pharmaceutical compositions can comprises excipients,including liquids such as water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. The pharmaceutical excipients canbe, for example, saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea and the like. In addition, auxiliary,stabilizing, thickening, lubricating, and coloring agents can be used.In one embodiment, the pharmaceutically acceptable excipients aresterile when administered to a subject. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

Dosage forms suitable for parenteral administration (e.g. subcutaneous,intradermal, subdermal, intramuscular, intravenous, intraperitoneal,intra-articular, and infusion) include, for example, solutions,suspensions, dispersions, emulsions, and the like. They may also bemanufactured in the form of sterile solid compositions (e.g. lyophilizedcomposition), which can be dissolved or suspended in sterile injectablemedium immediately before use. They may contain, for example, suspendingor dispersing agents known in the art.

In some embodiments, the formulations described herein may comprisealbumin and a nucleic acid molecule.

In some embodiments, the invention relates to a cosmetic composition. Inone embodiment, the cosmetic composition comprises albumin. In anotherembodiment, the albumin is treated with an ion-exchange resin orcharcoal. In yet another embodiment, the cosmetic composition comprisesa nucleic acid molecule. In a further embodiment, the cosmeticcomposition comprises both albumin and a nucleic acid molecule. Stillother embodiments are directed to a cosmetic treatment articlecomprising a cosmetic composition contained in a device configured todeliver the composition to a patient. Still other embodiments aredirected to a device configured to deliver a cosmetic composition to apatient. In one embodiment, the nucleic acid molecule encodes a memberof the group: elastin, collagen, tyrosinase, melanocortin 1 receptor,keratin, filaggren, an antibody, and hyaluronan synthase or abiologically active fragment, variant, analogue or family memberthereof.

In some embodiments, the present invention provides treatment regimens.The inventors have discovered that the doses and administrationdescribed herein can produce a substantial protein expression effectquickly (e.g. in about 6, or about 12, or about 24, or about 36, orabout 48 hours). Further, these effects can be sustained for about 7days, or longer. In some embodiments, the present methods provide foradministration of a nucleic acid drug, including RNA comprising one ormore non-canonical nucleotides, about weekly to about once every 20weeks.

In some embodiments, the nucleic acid drug, including RNA comprising oneor more non-canonical nucleotides, is administered about weekly, for atleast 2 weeks (e.g. 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 weeks).In some embodiments, the nucleic acid drug, including RNA comprising oneor more non-canonical nucleotides, is administered about every otherweek for at least one month (e.g. 1, or 2, or 3, or 4, or 5, or 6, or 12months). In some embodiments, the nucleic acid drug, including RNAcomprising one or more non-canonical nucleotides, is administeredmonthly or about every other month. In some embodiments, the nucleicacid drug, including RNA comprising one or more non-canonicalnucleotides, is administered is administered for at least two months, orat least 4 months, or at least 6 months, or at least 9 months, or atleast one year.

In some embodiments, the nucleic acid drug, including RNA comprising oneor more non-canonical nucleotides, is administered about weekly, orabout once every 2 weeks, or about once every 3 weeks, or about onceevery 4 weeks, or about once every 5 weeks, or about once every 6 weeks,or about once every 7 weeks, or about once every 8 weeks, or about onceevery 9 weeks, or about once every 10 weeks, or about once every 11weeks, or about once every 12 weeks, or about once every 13 weeks, orabout once every 14 weeks, or about once every 15 weeks, or about onceevery 20 weeks, or about once every 24 weeks.

In some embodiments, the nucleic acid drug, including RNA comprising oneor more non-canonical nucleotides, is administered no more than aboutweekly, or about once every 2 weeks, or about once every 3 weeks, orabout once every 4 weeks, or about once every 5 weeks, or about onceevery 6 weeks, or about once every 7 weeks, or about once every 8 weeks,or about once every 9 weeks, or about once every 10 weeks, or about onceevery 11 weeks, or about once every 12 weeks, or about once every 13weeks, or about once every 14 weeks, or about once every 15 weeks, orabout once every 20 weeks, or about 24 weeks.

Certain proteins have long half-lives, and can persist in tissues forseveral hours, days, weeks, months or years. It has now been discoveredthat certain methods of treating a patient can result in accumulation ofone or more proteins, including, for example, one or more beneficialproteins. Certain embodiments are therefore directed to a method fortreating a patient comprising delivering to a patient in a series ofdoses a nucleic acid encoding one or more proteins. In one embodimentthe nucleic acid comprises RNA comprising one or more non-canonicalnucleotides. In another embodiment, a first dose is given at a firsttime-point. In yet another embodiment, a second dose is given at asecond time-point. In a further embodiment, the amount of at least oneof the one or more proteins in the patient at the second time-point isgreater than the amount of said protein at the first time-point. In astill further embodiment, the method results in the accumulation of saidprotein in the patient.

In various embodiments, the present invention relates to nucleic aciddrugs, which, in various embodiments are RNA comprising one or morenon-canonical nucleotides. Certain non-canonical nucleotides, whenincorporated into RNA molecules, can reduce the toxicity of the RNAmolecules, in part, without wishing to be bound by theory, byinterfering with binding of proteins that detect exogenous nucleicacids, for example, protein kinase R, Rig-1 and the oligoadenylatesynthetase family of proteins. Non-canonical nucleotides that have beenreported to reduce the toxicity of RNA molecules when incorporatedtherein include: pseudouridine, 5-methyluridine, 2-thiouridine,5-methylcytidine, N6-methyladenosine, and certain combinations thereof.However, the chemical characteristics of non-canonical nucleotides thatcan enable them to lower the in vivo toxicity of RNA molecules have,until this point, remained unknown. Furthermore, incorporation of largeamounts of most non-canonical nucleotides, for example, 5-methyluridine,2-thiouridine, 5-methylcytidine, and N6-methyladenosine, can reduce theefficiency with which RNA molecules can be translated into protein,limiting the utility of RNA molecules containing these nucleotides inapplications that require protein expression. In addition, whilepseudouridine can be completely substituted for uridine in RNA moleculeswithout reducing the efficiency with which the synthetic RNA moleculescan be translated into protein, in certain situations, for example, whenperforming frequent, repeated transfections, synthetic RNA moleculescontaining only adenosine, guanosine, cytidine, and pseudouridine canexhibit excessive toxicity.

It has now been discovered that, and in some embodiments the inventionpertains to, RNA molecules containing one or more non-canonicalnucleotides that include one or more substitutions at the 2C and/or 4Cand/or 5C positions in the case of a pyrimidine or the 6C and/or 7Nand/or 8C positions in the case of a purine can be less toxic thansynthetic RNA molecules containing only canonical nucleotides, due inpart to the ability of substitutions at these positions to interferewith recognition of synthetic RNA molecules by proteins that detectexogenous nucleic acids, and furthermore, that substitutions at thesepositions can have minimal impact on the efficiency with which thesynthetic RNA molecules can be translated into protein, due in part tothe lack of interference of substitutions at these positions withbase-pairing and base-stacking interactions.

Examples of non-canonical nucleotides that include one or moresubstitutions at the 2C and/or 4C and/or 5C positions in the case of apyrimidine or the 6C and/or 7N and/or 8C positions in the case of apurine include, but are not limited to: 2-thiouridine, 5-azauridine,pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine,5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine,5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine,5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine,5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine,5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine,5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine,4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine,4-thio-5-aminouridine, 4-thio-5-hydroxyuridine,4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine,4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine,4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine,2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine,N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine,5-hydroxycytidine, 5-methoxycytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine,5-amino-5-azacytidine, 5-hydroxy-5-azacytidine,5-methylpseudoisocytidine, 5-aminopseudoisocytidine,5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine,N4-methylpseudoisocytidine, 2-thio-5-azacytidine,2-thiopseudoisocytidine, 2-thio-N4-methylcytidine,2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine,2-thio-5-methylcytidine, 2-thio-5-aminocytidine,2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine,2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine,2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine,2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine,2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine,N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine,N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine,N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine,N4-methyl-5-aminopseudoisocytidine,N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine,N4-aminopseudoisocytidine, N4-amino-5-methylcytidine,N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine,N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine,N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine,N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine,N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine,N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine,N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine,N4-hydroxy-5-methylpseudoisocytidine,N4-hydroxy-5-aminopseudoisocytidine,N4-hydroxy-5-hydroxypseudoisocytidine,2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine,2-thio-N4-methyl-5-hydroxycytidine,2-thio-N4-methyl-5-methyl-5-azacytidine,2-thio-N4-methyl-5-amino-5-azacytidine,2-thio-N4-methyl-5-hydroxy-5-azacytidine,2-thio-N4-methyl-5-methylpseudoisocytidine,2-thio-N4-methyl-5-aminopseudoisocytidine,2-thio-N4-methyl-5-hydroxypseudoisocytidine,2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine,2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine,2-thio-N4-amino-5-hydroxycytidine,2-thio-N4-amino-5-methyl-5-azacytidine,2-thio-N4-amino-5-amino-5-azacytidine,2-thio-N4-amino-5-hydroxy-5-azacytidine,2-thio-N4-amino-5-methylpseudoisocytidine,2-thio-N4-amino-5-aminopseudoisocytidine,2-thio-N4-amino-5-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine,2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine,2-thio-N4-hydroxy-5-hydroxycytidine,2-thio-N4-hydroxy-5-methyl-5-azacytidine,2-thio-N4-hydroxy-5-amino-5-azacytidine,2-thio-N4-hydroxy-5-hydroxy-5-azacytidine,2-thio-N4-hydroxy-5-methylpseudoisocytidine,2-thio-N4-hydroxy-5-aminopseudoisocytidine,2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine,N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine,8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine,7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine,N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine,N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine,N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine,N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine,8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine,7-deaza-8-azaguanosine, 6-thio-7-deaza-8-azaguanosine, and5-methoxyuridine.

In some embodiments, the invention relates to one or more non-canonicalnucleotides selected from 5-hydroxycytidine, 5-methylcytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine,5-methoxycytidine, 5-hydroxyuridine, 5-hydroxymethyluridine,5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine,5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridine. In some embodiments,at least 50%, or at least 55%, or at least 60%, or at least 65%, or atleast 70%, or at least 75%, or at least 80%, or at least 85%, or atleast 90%, or at least 95%, or 100% of the non-canonical nucleotides areone or more of 5-hydroxycytidine, 5-methylcytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine,5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine,5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine,5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine,5-methylpseudouridine, 5-hydroxymethylpseudouridine,5-carboxypseudouridine, 5-formylpseudouridine, and5-methoxypseudouridine.

In some embodiments, at least about 50%, or at least about 55%%, or atleast 60%, or at least 65%, or at least 70%, or at least 75%, or atleast 80%, or at least 85%, or at least 90%, or at least 95%, or 100% ofcytidine residues are non-canonical nucleotides selected from5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine.

In some embodiments, at least about 20%, or about 30%, or about 40%, orabout 50%, or at least about 55%, or at least 60%, or at least 65%, orat least 70%, or at least 75%, or at least 80%, or at least 85%, or atleast 90%, or at least 95%, or 100% of uridine residues arenon-canonical nucleotides selected from 5-hydroxyuridine,5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine,5-formyluridine, 5-methoxyuridine, pseudouridine,5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridine.

In some embodiments, at least about 10% (e.g. 10%, or about 20%, orabout 30%, or about 40%, or about 50%) of guanosine residues arenon-canonical nucleotides, and the non-canonical nucleotide isoptionally 7-deazaguanosine. In some embodiments, the RNA contains nomore than about 50% 7-deazaguanosine in place of guanosine residues.

In some embodiments, the RNA does not contain non-canonical nucleotidesin place of adenosine residues.

Note that alternative naming schemes exist for certain non-canonicalnucleotides. For example, in certain situations, 5-methylpseudouridinecan be referred to as “3-methylpseudouridine” or“N3-methylpseudouridine” or “1-methylpseudouridine” or“N1-methylpseudouridine”.

Nucleotides that contain the prefix “amino” can refer to any nucleotidethat contains a nitrogen atom bound to the atom at the stated positionof the nucleotide, for example, 5-aminocytidine can refer to5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly,nucleotides that contain the prefix “methyl” can refer to any nucleotidethat contains a carbon atom bound to the atom at the stated position ofthe nucleotide, for example, 5-methylcytidine can refer to5-methylcytidine, 5-ethylcytidine, and 5-hydroxymethylcytidine,nucleotides that contain the prefix “thio” can refer to any nucleotidethat contains a sulfur atom bound to the atom at the given position ofthe nucleotide, and nucleotides that contain the prefix “hydroxy” canrefer to any nucleotide that contains an oxygen atom bound to the atomat the given position of the nucleotide, for example, 5-hydroxyuridinecan refer to 5-hydroxyuridine and uridine with a methyl group bound toan oxygen atom, wherein the oxygen atom is bound to the atom at the 5Cposition of the uridine.

Certain embodiments are therefore directed to RNA comprising one or morenon-canonical nucleotides, wherein the RNA molecule contains one or morenucleotides that includes one or more substitutions at the 2C and/or 4Cand/or 5C positions in the case of a pyrimidine or the 6C and/or 7Nand/or 8C positions in the case of a purine. Other embodiments aredirected to a therapeutic, wherein the therapeutic contains one or moreRNA molecules comprising one or more non-canonical nucleotides, andwherein the one or more RNA molecules comprising one or morenon-canonical nucleotides contains one or more nucleotides that includesone or more substitutions at the 2C and/or 4C and/or 5C positions in thecase of a pyrimidine or the 6C and/or 7N and/or 8C positions in the caseof a purine. In one embodiment, the therapeutic comprises a transfectionreagent. In another embodiment, the transfection reagent comprises acationic lipid, liposome or micelle. In still another embodiment, theliposome or micelle comprises folate and the therapeutic composition hasanti-cancer activity. In another embodiment, the one or more nucleotidesincludes at least one of pseudouridine, 2-thiouridine, 4-thiouridine,5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine,2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine,5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, 5-methylpseudoisocytidine, 7-deazaadenosine,7-deazaguanosine, 6-thioguanosine, and 6-thio-7-deazaguanosine. Inanother embodiment, the one or more nucleotides includes at least one ofpseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine,5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, and5-aminopseudouridine and at least one of pseudoisocytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, and 5-methylpseudoisocytidine. In stillanother embodiment, the one or more nucleotides include at least one ofpseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine,5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, and 5-methylpseudouridine,5-aminopseudouridine and at least one of pseudoisocytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine,2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine,5-aminopseudoisocytidine, and 5-methylpseudoisocytidine and at least oneof 7-deazaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, and5-methoxyuridine. In yet another embodiment, the one or more nucleotidesincludes: 5-methylcytidine and 7-deazaguanosine. In another embodiment,the one or more nucleotides also includes pseudouridine or 4-thiouridineor 5-methyluridine or 5-aminouridine or 4-thiopseudouridine or5-methylpseudouridine or 5-aminopseudouridine. In a still anotherembodiment, the one or more nucleotides also includes 7-deazaadenosine.In another embodiment, the one or more nucleotides includes:pseudoisocytidine and 7-deazaguanosine and 4-thiouridine. In yet anotherembodiment, the one or more nucleotides includes: pseudoisocytidine or7-deazaguanosine and pseudouridine. In still another embodiment, the oneor more nucleotides includes: 5-methyluridine and 5-methylcytidine and7-deazaguanosine. In a further embodiment, the one or more nucleotidesincludes: pseudouridine or 5-methylpseudouridine and 5-methylcytidineand 7-deazaguanosine. In another embodiment, the one or more nucleotidesincludes: pseudoisocytidine and 7-deazaguanosine and pseudouridine. Inone embodiment, the RNA comprising one or more non-canonical nucleotidesis present in vivo.

Certain non-canonical nucleotides can be incorporated more efficientlythan other non-canonical nucleotides into RNA molecules by RNApolymerases that are commonly used for in vitro transcription, due inpart to the tendency of these certain non-canonical nucleotides toparticipate in standard base-pairing interactions and base-stackinginteractions, and to interact with the RNA polymerase in a mannersimilar to that in which the corresponding canonical nucleotideinteracts with the RNA polymerase. As a result, certain nucleotidemixtures containing one or more non-canonical nucleotides can bebeneficial in part because in vitro-transcription reactions containingthese nucleotide mixtures can yield a large quantity of RNA. Certainembodiments are therefore directed to a nucleotide mixture containingone or more nucleotides that includes one or more substitutions at the2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6Cand/or 7N and/or 8C positions in the case of a purine. Nucleotidemixtures include, but are not limited to (numbers preceding eachnucleotide indicate an exemplary fraction of the non-canonicalnucleotide triphosphate in an in vitro-transcription reaction, forexample, 0.2 pseudoisocytidine refers to a reaction containingadenosine-5′-triphosphate, guanosine-5′-triphosphate,uridine-5′-triphosphate, cytidine-5′-triphosphate, andpseudoisocytidine-5′-triphosphate, wherein pseudoisocytidine-5′-triphosphate is present in the reaction at an amount approximately equal to0.2 times the total amount ofpseudoisocytidine-5′-triphosphate+cytidine-5′-triphosphate that ispresent in the reaction, with amounts measured either on a molar or massbasis, and wherein more than one number preceding a nucleoside indicatesa range of exemplary fractions): 1.0 pseudouridine, 0.1-0.82-thiouridine, 0.1-0.8 5-methyluridine, 0.2-1.0 5-hydroxyuridine,0.2-1.0 5-methoxyuridine, 0.1-1.0 5-aminouridine, 0.1-1.0 4-thiouridine,0.1-1.0 2-thiopseudouridine, 0.1-1.0 4-thiopseudouridine, 0.1-1.05-hydroxypseudouridine, 0.2-1 5-methylpseudouridine, 0.2-1.05-methoxypseudouridine, 0.1-1.0 5-aminopseudouridine, 0.2-1.02-thiocytidine, 0.1-0.8 pseudoisocytidine, 0.2-1.0 5-methylcytidine,0.2-1.0 5-hydroxycytidine, 0.2-1.0 5-hydroxymethylcytidine, 0.2-1.05-methoxycytidine, 0.1-1.0 5-aminocytidine, 0.2-1.0 N4-methylcytidine,0.2-1.0 5-methylpseudoisocytidine, 0.2-1.0 5-hydroxypseudoisocytidine,0.2-1.0 5-aminopseudoisocytidine, 0.2-1.0 N4-methylpseudoisocytidine,0.2-1.0 2-thiopseudoisocytidine, 0.2-1.0 7-deazaguanosine, 0.2-1.06-thioguanosine, 0.2-1.0 6-thio-7-deazaguanosine, 0.2-1.08-azaguanosine, 0.2-1.0 7-deaza-8-azaguanosine, 0.2-1.06-thio-8-azaguanosine, 0.1-0.5 7-deazaadenosine, and 0.1-0.5N6-methyladenosine.

In various embodiments, the RNA comprising one or more non-canonicalnucleotides composition or synthetic polynucleotide composition (e.g.,which may be prepared by in vitro transcription) contains substantiallyor entirely the canonical nucleotide at positions having adenine or “A”in the genetic code. The term “substantially” in this context refers toat least 90%. In these embodiments, the RNA composition or syntheticpolynucleotide composition may further contain (e.g., consist of)7-deazaguanosine at positions with “G” in the genetic code as well asthe corresponding canonical nucleotide “G”, and the canonical andnon-canonical nucleotide at positions with G may be in the range of 5:1to 1:5, or in some embodiments in the range of 2:1 to 1:2. In theseembodiments, the RNA composition or synthetic polynucleotide compositionmay further contain (e.g., consist of) one or more (e.g., two, three orfour) of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine at positions with“C” in the genetic code as well as the canonical nucleotide “C”, and thecanonical and non-canonical nucleotide at positions with C may be in therange of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2.In some embodiments, the level of non-canonical nucleotide at positionsof “C” are as described in the preceding paragraph. In theseembodiments, the RNA composition or synthetic polynucleotide compositionmay further contain (e.g., consist of) one or more (e.g., two, three, orfour) of 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine,5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine,5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridineat positions with “U”in the genetic code as well as the canonical nucleotide “U”, and thecanonical and non-canonical nucleotide at positions with “U” may be inthe range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to1:2. In some embodiments, the level of non-canonical nucleotide atpositions of “U” are as described in the preceding paragraph.

It has now been discovered that combining certain non-canonicalnucleotides can be beneficial in part because the contribution ofnon-canonical nucleotides to lowering the toxicity of RNA molecules canbe additive. Certain embodiments are therefore directed to a nucleotidemixture, wherein the nucleotide mixture contains more than one of thenon-canonical nucleotides listed above, for example, the nucleotidemixture contains both pseudoisocytidine and 7-deazaguanosine or thenucleotide mixture contains both N4-methylcytidine and 7-deazaguanosine,etc. In one embodiment, the nucleotide mixture contains more than one ofthe non-canonical nucleotides listed above, and each of thenon-canonical nucleotides is present in the mixture at the fractionlisted above, for example, the nucleotide mixture contains 0.1-0.8pseudoisocytidine and 0.2-1.0 7-deazaguanosine or the nucleotide mixturecontains 0.2-1.0 N4-methylcytidine and 0.2-1.0 7-deazaguanosine, etc.

In certain situations, for example, when it may not be necessary ordesirable to maximize the yield of an in vitro-transcription reaction,nucleotide fractions other than those given above may be used. Theexemplary fractions and ranges of fractions listed above relate tonucleotide-triphosphate solutions of typical purity (greater than 90%purity). Larger fractions of these and other nucleotides can be used byusing nucleotide-triphosphate solutions of greater purity, for example,greater than about 95% purity or greater than about 98% purity orgreater than about 99% purity or greater than about 99.5% purity, whichcan be achieved, for example, by purifying the nucleotide triphosphatesolution using existing chemical-purification technologies such ashigh-pressure liquid chromatography (HPLC) or by other means. In oneembodiment, nucleotides with multiple isomers are purified to enrich thedesired isomer.

Other embodiments are directed to a method for inducing a cell in vivoto express a protein of interest by contacting the cell with a RNAmolecule that contains one or more non-canonical nucleotides thatincludes one or more substitutions at the 2C and/or 4C and/or 5Cpositions in the case of a pyrimidine or the 6C and/or 7N and/or 8Cpositions in the case of a purine. Still other embodiments are directedto a method for transfecting, reprogramming, and/or gene-editing a cellin vivo by contacting the cell with a RNA molecule that contains one ormore non-canonical nucleotides that includes one or more substitutionsat the 2C and/or 4C and/or 5C positions in the case of a pyrimidine orthe 6C and/or 7N and/or 8C positions in the case of a purine. In oneembodiment, the RNA molecule is produced by in vitro transcription. Inone embodiment, the RNA molecule encodes one or more reprogrammingfactors. In another embodiment, the one or more reprogramming factorsincludes Oct4 protein. In another embodiment, the cell is also contactedwith a RNA molecule that encodes Sox2 protein. In yet anotherembodiment, the cell is also contacted with a RNA molecule that encodesKlf4 protein. In yet another embodiment, the cell is also contacted witha RNA molecule that encodes c-Myc protein. In yet another embodiment,the cell is also contacted with a RNA molecule that encodes Lin28protein.

Enzymes such as T7 RNA polymerase may preferentially incorporatecanonical nucleotides in an in vitro-transcription reaction containingboth canonical and non-canonical nucleotides. As a result, an invitro-transcription reaction containing a certain fraction of anon-canonical nucleotide may yield RNA containing a different, oftenlower, fraction of the non-canonical nucleotide than the fraction atwhich the non-canonical nucleotide was present in the reaction. Incertain embodiments, references to nucleotide incorporation fractions(for example, “a synthetic RNA molecule containing 50%pseudoisocytidine” or “0.1-0.8 pseudoisocytidine”) therefore can referboth to RNA molecules containing the stated fraction of the nucleotide,and to RNA molecules synthesized in a reaction containing the statedfraction of the nucleotide (or nucleotide derivative, for example,nucleotide-triphosphate), even though such a reaction may yield RNAcontaining a different fraction of the nucleotide than the fraction atwhich the non-canonical nucleotide was present in the reaction.

Different nucleotide sequences can encode the same protein by utilizingalternative codons. In certain embodiments, references to nucleotideincorporation fractions therefore can refer both to RNA moleculescontaining the stated fraction of the nucleotide, and to RNA moleculesencoding the same protein as a different RNA molecule, wherein thedifferent RNA molecule contains the stated fraction of the nucleotide.

Certain embodiments are directed to a kit containing one or morematerials needed to practice the present invention. In one embodiment,the kit contains one or more synthetic RNA molecules. In one embodiment,the kit contains one or more synthetic RNA molecules that encode one ormore reprogramming factors and/or gene-editing proteins. In anotherembodiment, the one or more synthetic RNA molecules contain one or morenon-canonical nucleotides that include one or more substitutions at the2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6Cand/or 7N and/or 8C positions in the case of a purine. In anotherembodiment, the kit contains one or more of: a transfection medium, atransfection reagent, a complexation medium, and a matrix solution. Inone embodiment, the matrix solution contains fibronectin and/orvitronectin or recombinant fibronectin and/or recombinant vitronectin.In one embodiment, one or more of the components of the kit are presentas a plurality of aliquots. In one embodiment, the kit contains aliquotsof nucleic acid transfection-reagent complexes. In another embodiment,the kit contains aliquots of nucleic acid transfection-reagent complexesthat are provided in a solid form, for example, as frozen orfreeze-dried pellets. In yet another embodiment, the kit containsaliquots of medium, wherein each aliquot contains transfectionreagent-nucleic acid complexes that are stabilized either by chemicaltreatment or by freezing.

Transfection, in general, and reprogramming, in particular, can bedifficult and time-consuming techniques that can be repetitive and proneto error. However, these techniques are often performed manually due tothe lack of automated transfection equipment. Certain embodiments aretherefore directed to a system that can transfect, reprogram, and/orgene-edit cells in vivo in an automated or semi-automated manner.

It has now been discovered that the non-canonical nucleotide members ofthe 5-methylcytidine de-methylation pathway, when incorporated intosynthetic RNA, can increase the efficiency with which the synthetic RNAcan be translated into protein in vivo, and can decrease the toxicity ofthe synthetic RNA in vivo. These non-canonical nucleotides include, forexample: 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine,and 5-carboxycytidine (a.k.a. “cytidine-5-carboxylic acid”). Certainembodiments are therefore directed to a nucleic acid. In someembodiments, the nucleic acid is present in vivo. In one embodiment, thenucleic acid is a synthetic RNA molecule. In another embodiment, thenucleic acid comprises one or more non-canonical nucleotides. In oneembodiment, the nucleic acid comprises one or more non-canonicalnucleotide members of the 5-methylcytidine de-methylation pathway. Inanother embodiment, the nucleic acid comprises at least one of:5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and5-carboxycytidine or a derivative thereof. In a further embodiment, thenucleic acid comprises at least one of: pseudouridine,5-methylpseudouridine, 5-hydroxyuridine, 5-methyluridine,5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine,N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof.

5-Methylcytidine De-Methylation Pathway

Certain embodiments are directed to a protein. Other embodiments aredirected to a nucleic acid that encodes a protein. In one embodiment,the protein is a protein of interest. In another embodiment, the proteinis selected from: a reprogramming protein and a gene-editing protein. Inone embodiment, the nucleic acid is a plasmid. In another embodiment,the nucleic acid is present in a virus or viral vector. In a furtherembodiment, the virus or viral vector is replication incompetent. In astill further embodiment, the virus or viral vector is replicationcompetent. In one embodiment, the virus or viral vector includes atleast one of: an adenovirus, a retrovirus, a lentivirus, a herpes virus,an adeno-associated virus or a natural or engineered variant thereof,and an engineered virus.

It has also been discovered that certain combinations of non-canonicalnucleotides can be particularly effective at increasing the efficiencywith which synthetic RNA can be translated into protein in vivo, anddecreasing the toxicity of synthetic RNA in vivo, for example, thecombinations: 5-methyluridine and 5-methylcytidine, 5-hydroxyuridine and5-methylcytidine, 5-hydroxyuridine and 5-hydroxymethylcytidine,5-methyluridine and 7-deazaguanosine, 5-methylcytidine and7-deazaguanosine, 5-methyluridine, 5-methylcytidine, and7-deazaguanosine, and 5-methyluridine, 5-hydroxymethylcytidine, and7-deazaguanosine. Certain embodiments are therefore directed to anucleic acid comprising at least two of: 5-methyluridine,5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or oneor more derivatives thereof. Other embodiments are directed to a nucleicacid comprising at least three of: 5-methyluridine, 5-methylcytidine,5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivativesthereof. Other embodiments are directed to a nucleic acid comprising allof: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and7-deazaguanosine or one or more derivatives thereof. In one embodiment,the nucleic acid comprises one or more 5-methyluridine residues, one ormore 5-methylcytidine residues, and one or more 7-deazaguanosineresidues or one or more 5-methyluridine residues, one or more5-hydroxymethylcytidine residues, and one or more 7-deazaguanosineresidues.

It has been further discovered that synthetic RNA molecules containingcertain fractions of certain non-canonical nucleotides and combinationsthereof can exhibit particularly high translation efficiency and lowtoxicity in vivo. Certain embodiments are therefore directed to anucleic acid comprising at least one of: one or more uridine residues,one or more cytidine residues, and one or more guanosine residues, andcomprising one or more non-canonical nucleotides. In one embodiment,between about 20% and about 80% of the uridine residues are5-methyluridine residues. In another embodiment, between about 30% andabout 50% of the uridine residues are 5-methyluridine residues. In afurther embodiment, about 40% of the uridine residues are5-methyluridine residues. In one embodiment, between about 60% and about80% of the cytidine residues are 5-methylcytidine residues. In anotherembodiment, between about 80% and about 100% of the cytidine residuesare 5-methylcytidine residues. In a further embodiment, about 100% ofthe cytidine residues are 5-methylcytidine residues. In a still furtherembodiment, between about 20% and about 100% of the cytidine residuesare 5-hydroxymethylcytidine residues. In one embodiment, between about20% and about 80% of the guanosine residues are 7-deazaguanosineresidues. In another embodiment, between about 40% and about 60% of theguanosine residues are 7-deazaguanosine residues. In a furtherembodiment, about 50% of the guanosine residues are 7-deazaguanosineresidues. In one embodiment, between about 20% and about 80% or betweenabout 30% and about 60% or about 40% of the cytidine residues areN4-methylcytidine and/or N4-acetylcytidine residues. In anotherembodiment, each cytidine residue is a 5-methylcytidine residue. In afurther embodiment, about 100% of the cytidine residues are5-methylcytidine residues and/or 5-hydroxymethylcytidine residues and/orN4-methylcytidine residues and/or N4-acetylcytidine residues and/or oneor more derivatives thereof. In a still further embodiment, about 40% ofthe uridine residues are 5-methyluridine residues, between about 20% andabout 100% of the cytidine residues are N4-methylcytidine and/orN4-acetylcytidine residues, and about 50% of the guanosine residues are7-deazaguanosine residues. In one embodiment, about 40% of the uridineresidues are 5-methyluridine residues and about 100% of the cytidineresidues are 5-methylcytidine residues. In another embodiment, about 40%of the uridine residues are 5-methyluridine residues and about 50% ofthe guanosine residues are 7-deazaguanosine residues. In a furtherembodiment, about 100% of the cytidine residues are 5-methylcytidineresidues and about 50% of the guanosine residues are 7-deazaguanosineresidues. In a further embodiment, about 100% of the uridine residuesare 5-hydroxyuridine residues. In one embodiment, about 40% of theuridine residues are 5-methyluridine residues, about 100% of thecytidine residues are 5-methylcytidine residues, and about 50% of theguanosine residues are 7-deazaguanosine residues. In another embodiment,about 40% of the uridine residues are 5-methyluridine residues, betweenabout 20% and about 100% of the cytidine residues are5-hydroxymethylcytidine residues, and about 50% of the guanosineresidues are 7-deazaguanosine residues. In some embodiments, less than100% of the cytidine residues are 5-methylcytidine residues. In otherembodiments, less than 100% of the cytidine residues are5-hydroxymethylcytidine residues. In one embodiment, each uridineresidue in the synthetic RNA molecule is a pseudouridine residue or a5-methylpseudouridine residue. In another embodiment, about 100% of theuridine residues are pseudouridine residues and/or 5-methylpseudouridineresidues. In a further embodiment, about 100% of the uridine residuesare pseudouridine residues and/or 5-methylpseudouridine residues, about100% of the cytidine residues are 5-methylcytidine residues, and about50% of the guanosine residues are 7-deazaguanosine residues.

Other non-canonical nucleotides that can be used in place of or incombination with 5-methyluridine include, but are not limited to:pseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine,5-methoxyuridine, 5-methoxypseudouridine, 5-carboxyuridine,5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine,5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, and5-methylpseudouridine (a.k.a. “1-methylpseudouridine”, a.k.a.“N1-methylpseudouridine”) or one or more derivatives thereof. Othernon-canonical nucleotides that can be used in place of or in combinationwith 5-methylcytidine and/or 5-hydroxymethylcytidine include, but arenot limited to: pseudoisocytidine, 5-methylpseudoisocytidine,5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine,5-methoxycytidine, N4-methylcytidine, N4-acetylcytidine or one or morederivatives thereof. In certain embodiments, for example, whenperforming only a single transfection, injection or delivery or when thecells, tissue, organ or patient being transfected, injected or deliveredto are not particularly sensitive to transfection-associated toxicity orinnate-immune signaling, the fractions of non-canonical nucleotides canbe reduced. Reducing the fraction of non-canonical nucleotides can bebeneficial, in part, because reducing the fraction of non-canonicalnucleotides can reduce the cost of the nucleic acid. In certainsituations, for example, when minimal immunogenicity of the nucleic acidis desired, the fractions of non-canonical nucleotides can be increased.

Enzymes such as T7 RNA polymerase may preferentially incorporatecanonical nucleotides in an in vitro-transcription reaction containingboth canonical and non-canonical nucleotides. As a result, an invitro-transcription reaction containing a certain fraction of anon-canonical nucleotide may yield RNA containing a different, oftenlower, fraction of the non-canonical nucleotide than the fraction atwhich the non-canonical nucleotide was present in the reaction. Incertain embodiments, references to nucleotide incorporation fractions(for example, “50% 5-methyluridine”) therefore can refer both to nucleicacids containing the stated fraction of the nucleotide, and to nucleicacids synthesized in a reaction containing the stated fraction of thenucleotide (or nucleotide derivative, for example,nucleotide-triphosphate), even though such a reaction may yield anucleic acid containing a different fraction of the nucleotide than thefraction at which the non-canonical nucleotide was present in thereaction. In addition, different nucleotide sequences can encode thesame protein by utilizing alternative codons. In certain embodiments,references to nucleotide incorporation fractions therefore can referboth to nucleic acids containing the stated fraction of the nucleotide,and to nucleic acids encoding the same protein as a different nucleicacid, wherein the different nucleic acid contains the stated fraction ofthe nucleotide.

Certain embodiments are directed to a nucleic acid comprising a 5′-capstructure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivativethereof. In one embodiment, the nucleic acid comprises one or more UTRs.In another embodiment, the one or more UTRs increase the stability ofthe nucleic acid. In a further embodiment, the one or more UTRs comprisean alpha-globin or beta-globin 5′-UTR. In a still further embodiment,the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. Ina still further embodiment, the synthetic RNA molecule comprises analpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that issubstantially similar to the Kozak consensus sequence. In anotherembodiment, the nucleic acid comprises a 3′-poly(A) tail. In a furtherembodiment, the 3′-poly(A) tail is between about 20 nt and about 250 ntor between about 120 nt and about 150 nt long. In a further embodiment,the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, orabout 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt,or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, orabout 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, orabout 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

Certain embodiments are directed to methods of making nucleic aciddrugs, including RNA comprising one or more non-canonical nucleotides.Such methods yield substantially stable RNA.

In various embodiments, the present methods and compositions find use inmethods of treating, preventing or ameliorating a disease, disorderand/or condition. For instance, in some embodiments, the describedmethods of in vivo delivery, including various effective doses,administration strategies and formulations are used in a method oftreatment.

In various embodiments, the present methods and compositions find use inmethods of altering, modifying and/or changing a tissue (e.g.cosmetically).

In various embodiments, the present methods and compositions includeusing a nucleic acid drug, including a synthetic RNA, in the diagnosing,treating, preventing or ameliorating of a disease, disorder and/orcondition described herein. In various embodiments, the present methodsand compositions include using a nucleic acid drug, including asynthetic RNA, in the altering, modifying and/or changing of a tissue(e.g. cosmetically).

Generally speaking, in various embodiments, a synthetic RNA as describedherein is administered to a human at specific doses described herein andthe synthetic RNA comprises a sequence, sometimes referred to as atarget sequence that encodes a protein of interest, which may be atherapeutic protein.

Synthetic RNA comprising only canonical nucleotides can bind to patternrecognition receptors, can be recognized as a pathogen-associatedmolecular pattern, and can trigger a potent immune response in cells,which can result in translation block, the secretion of inflammatorycytokines, and cell death. It has now been discovered that synthetic RNAcomprising certain non-canonical nucleotides can evade detection by theinnate immune system, and can be translated at high efficiency intoprotein, including in humans. It has been further discovered thatsynthetic RNA comprising at least one of the non-canonical nucleotidesdescribed herein, including, for example, a member of the group:5-methylcytidine, 5-hydroxycytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine,5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine,5-carboxyuridine, 5-methoxyuridine, 5-formyluridine,5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-methoxypseudouridine, and 5-formylpseudouridine can evade detection bythe innate immune system, and can be translated at high efficiency intoprotein, including in humans. Certain embodiments are therefore directedto a method for inducing a cell to express a protein of interestcomprising contacting a cell with synthetic RNA. Other embodiments aredirected to a method for transfecting a cell with synthetic RNAcomprising contacting a cell with a solution comprising one or moresynthetic RNA molecules. Still other embodiments are directed to amethod for treating a patient comprising administering to the patientsynthetic RNA. In one embodiment, the synthetic RNA comprises at leastone of the non-canonical nucleotides described herein, including, forexample, a member of the group: 5-methylcytidine, 5-hydroxycytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine,5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine,5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine,5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-methoxypseudouridine, and 5-formylpseudouridine. In anotherembodiment, the synthetic RNA encodes a protein of interest. ExemplaryRNAs may contain combinations and levels of non-canonical andnon-canonical nucleotides as described elsewhere herein, including withrespect to the expression of any protein of interest described herein.In yet another embodiment, the method results in the expression of theprotein of interest. In a further embodiment, the method results in theexpression of the protein of interest in the patient's skin.

Other embodiments are directed to a method for delivering a nucleic acidto a cell in vivo. Still other embodiments are directed to a method forinducing a cell in vivo to express a protein of interest. Still otherembodiments are directed to a method for treating a patient. In oneembodiment, the method comprises disrupting the stratum corneum. Inanother embodiment, the method comprises contacting a cell with anucleic acid. In yet another embodiment, the method results in the cellinternalizing the nucleic acid. In a further embodiment, the methodresults in the cell expressing the protein of interest. In a stillfurther embodiment, the method results in the expression of the proteinof interest in the patient. In a still further embodiment, the methodresults in the amelioration of one or more of the patient's symptoms. Ina still further embodiment, the patient is in need of the protein ofinterest. In a still further embodiment, the patient is deficient in theprotein of interest.

Still other embodiments are directed to a method for treating a patientcomprising delivering to a patient a composition. In one embodiment, thecomposition comprises albumin that is treated with an ion-exchange resinor charcoal. In another embodiment, the composition comprises one ormore nucleic acid molecules. In yet another embodiment, at least one ofthe one or more nucleic acid molecules encodes a protein of interest. Inone embodiment, the method results in the expression of the protein inthe patient's skin. In another embodiment, the method results in theexpression of a therapeutically or cosmetically effective amount of theprotein of interest in the patient. In yet another embodiment, themethod comprises administering a steroid. In a further embodiment, thesteroid is a member of the group: hydrocortisone and dexamethasone.

Some embodiments are directed to a therapeutic composition and/ormethods of treatment comprising a nucleic acid molecule encoding one ormore proteins, wherein at least one of the one or more proteins is anextracellular matrix protein. Still other embodiments are directed to acosmetic composition comprising a nucleic acid molecule encoding one ormore proteins, wherein at least one of the one or more proteins is anextracellular matrix protein.

Pigmentation disorders can cause severe symptoms in patients. It has nowbeen discovered that pigmentation disorders can be treated by deliveringto a patient a nucleic acid encoding tyrosinase. Certain embodiments aretherefore directed to a method for treating a pigmentation disorder.Other embodiments are directed to a method for altering the pigmentationof a patient. In one embodiment, the method comprises delivering to apatient a nucleic acid encoding tyrosinase. Other embodiments aredirected to a cosmetic composition comprising a nucleic acid encodingtyrosinase. Still other embodiments are directed to a therapeuticcomposition comprising a nucleic acid encoding tyrosinase. Still otherembodiments are directed to a method for increasing the ultravioletabsorption of a patient's skin. In one embodiment the method comprisesdelivering to a patient a nucleic acid encoding tyrosinase. In anotherembodiment, the method results in an increase in the ultravioletabsorption of the patient's skin. Still other embodiments are directedto a method for reducing photodamage to a person's skin upon exposure toultraviolet light. In one embodiment, the method results in thereduction of photodamage to the person's skin upon exposure toultraviolet light. Still other embodiments are directed to a method fortreating xeroderma pigmentosum. In one embodiment, the method comprisesdelivering to a patient a nucleic acid encoding tyrosinase. Still otherembodiments are directed to a method for treating epidermolysis bullosa.In one embodiment, the method comprises delivering to a patient anucleic acid encoding one or more of keratin 5, keratin 14, plectin, anintegrin family member, laminin, a laminin subunit, collagen XVII,collagen VII or a biologically active fragment, variant, analogue orfamily-member thereof. In one embodiment, the method comprisesdelivering to a patient a nucleic acid encoding collagen type VII. Inanother embodiment, the method comprises delivering to a patient anucleic acid encoding melanocortin 1 receptor. Still other embodimentsare directed to a method for treating xerosis. In one embodiment, themethod comprises delivering to a patient a nucleic acid encoding ahyaluronan synthase. In another embodiment, the patient is diagnosedwith atopic dermatitis. In yet another embodiment, the patient isdiagnosed with ichthyosis. Certain embodiments are directed to a methodfor treating a cosmetic condition. Other embodiments are directed to amethod for inducing tissue healing. In one embodiment, the methodcomprises delivering to a patient a nucleic acid encoding a hyaluronansynthase. In another embodiment, the cosmetic condition is a member ofthe group: wrinkles, sagging skin, thin skin, discoloration, and dryskin. In yet another embodiment, the patient has had cataract surgery.In some embodiments, the nucleic acid is synthetic RNA. In otherembodiments, the method results in the amelioration of one or more ofthe patient's symptoms. Other embodiments are directed to a method fortreating an indication by delivering to a cell or a patient a nucleicacid encoding a protein or a peptide. Still other embodiments aredirected to a composition comprising a nucleic acid encoding a proteinor a peptide. Indications that can be treated using the methods andcompositions of the present invention and proteins and peptides that canbe encoded by compositions of the present invention are set forth inTable 2A and/or Table 2B, and are given by way of example, and not byway of limitation. In one embodiment, the indication is selected fromTable 2A and/or Table 2B. In another embodiment the protein or peptideis selected from Table 2A and/or Table 2B. In yet another embodiment,the indication and the protein or peptide are selected from the same rowof Table 2A and/or Table 2B. In a further embodiment, the protein ofinterest is a member of the group: UCP1, UCP2, and UCP3. Otherembodiments are directed to methods for inducing a cell to express aplurality of proteins of interest. In one embodiment, the proteins ofinterest include at least two members of the group: a lipase, UCP1,UCP2, and UCP3. In another embodiment, the proteins of interest includea lipase and a member of the group: UCP1, UCP2, and UCP3. In anotherembodiment, the protein is a gene-editing protein. In yet anotherembodiment, the gene-editing protein targets a gene that is at leastpartly responsible for a disease phenotype. In yet another embodiment,the gene-editing protein targets a gene that encodes a protein selectedfrom Table 2A and/or Table 2B. In still another embodiment, thegene-editing protein corrects or eliminates, either alone or incombination with one or more other molecules or gene-editing proteins, amutation that is at least partly responsible for a disease phenotype.

In various embodiments, the present invention contemplates the targetingof the precursor forms and/or mature forms and/or isoforms and/ormutants of any of the proteins disclosed in Table 2A and/or Table 2B andsuch proteins. In some embodiments, any of the precursor forms and/ormature forms and/or isoforms and/or mutants have enhanced secretionrelative to the corresponding wild type proteins. In some embodiments,any of the precursor forms and/or mature forms and/or isoforms and/ormutants have altered half-lives (e.g. serum, plasma, intracellular)—forinstance, longer or shorter half-lives. In some embodiments, this isrelative to wild type.

TABLE 2A Illustrative Indications Illustrative Indication IllustrativeProtein/Peptide Acne Retinol Dehydrogenase 10 Aging Elastinsp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) Aging CollagenType I P02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO: 487)P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488) AgingCollagen Type III P02461|CO3A1_HUMAN Collagen alpha-1(III) chain(isoform 1) (SEQ ID NO: 489) Aging Collagen Type VII Q02388|CO7A1_HUMANCollagen alpha-1(VII) chain (SEQ ID NO: 490) Aging Hyaluronan SynthaseAging Telomerase Reverse Transcriptase Albinism TyrosinaseP14679|TYRO_HUMAN Tyrosinase (isoform 1) (SEQ ID NO: 491) AlportSyndrome Collagen Type IV P02462|CO4A1_HUMAN Collagen alpha-1(IV) chain(isoform 1) (SEQ ID NO: 492) P08572|CO4A2_HUMAN Collagen alpha-2(IV)chain (SEQ ID NO: 493) Q01955|CO4A3_HUMAN Collagen alpha-3(IV) chain(isoform 1) (SEQ ID NO: 494) P53420|CO4A4_HUMAN Collagen alpha-4(IV)chain (SEQ ID NO: 495) P29400|CO4A5_HUMAN Collagen alpha-5(IV) chain(isoform 1) (SEQ ID NO: 496) Q14031|CO4A6_HUMAN Collagen alpha-6(IV)(isoform A) (SEQ ID NO: 497) Anemia Erythropoietin Atopic DermatitisFilaggrin Cutis Laxa Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3)(SEQ ID NO: 486) Dry Skin Filaggrin Dystrophic Epidermolysis BullosaCollagen Type VII Q02388|CO7A1_HUMAN Collagen alpha-1(VII) chain (SEQ IDNO: 498) Ehlers-Danlos Syndrome Collagen Type V P20908|CO5A1_HUMANCollagen alpha-1(V) chain (SEQ ID NO: 499) P05997|CO5A2_HUMAN Collagenalpha-2(V) chain (SEQ ID NO: 500) P25940|CO5A3_HUMAN Collagen alpha-3(V)chain (SEQ ID NO: 501) Ehlers-Danlos Syndrome Collagen Type IP02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO: 487)P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488)Epidermolysis bullosa, lethal acantholytic ADAM17 P78536|ADA17_HUMANDisintegrin and metalloproteinase domain-containing protein 17 (isoformA) (SEQ ID NO: 502) Epidermolysis bullosa, type IV Collagen Type IIIP02461|CO3A1_HUMAN Collagen alpha-1(III) chain (isoform 1) (SEQ ID NO:489) Erythropoietic Protoporphyria Ferrochelatase P22830|HEMH_HUMANFerrochelatase, mitochondrial (isoform 1) (SEQ ID NO: 503) EczemaFilaggrin Excess Fat Thermogenin P25874|UCP1_HUMAN Mitochondrial brownfat uncoupling protein 1 (SEQ ID NO: 504) Excess Fat Lipase Lipoproteinlipase P06858|LIPL_HUMAN Lipoprotein lipase (SEQ ID NO: 516) Hepaticlipase P11150|LIPC_HUMAN Hepatic triacylglycerol lipase (SEQ ID NO: 517)Pancreatic lipase P16233|LIPP_HUMAN Pancreatic triacylglycerol lipase(SEQ ID NO: 518) Endothelial lipase (isoform 1) Q9Y5X9|LIPE_HUMANEndothelial lipase (SEQ ID NO: 519) Lysosomal lipase P38571|LICH_HUMANLysosomal acid lipase/cholesteryl ester hydrolase (isoform 1) (SEQ IDNO: 520) Hormone sensitive lipase Q05469|LIPS_HUMAN Hormone-sensitivelipas (isoform 1) (SEQ ID NO: 521) Gastric lipase P07098|LIPG_HUMANGastric triacylglycerol lipase (isoform 1) (SEQ ID NO: 522) PancreaticLipase-Related Protein 1) P54315|LIPR1_HUMAN Inactive pancreatic lipase-related protein 1 (isoform 1) (SEQ ID NO: 523) Pancreatic Lipase-RelatedProtein 2 P54317|LIPR2_HUMAN Pancreatic lipase-related protein 2 (SEQ IDNO: 524) Carboxyl Ester Lipase P19835|CEL_HUMAN Bile salt-activatedlipase (isoform long) (SEQ ID NO: 525) Hypotrichosis ADAM17P78536|ADA17_HUMAN Disintegrin and metalloproteinase domain-containingprotein 17 (isoform A) (SEQ ID NO: 502) Ichthyosis Vulgaris FilaggrinInfections Genetic Antibiotics (e.g. Anti-Sigma Factors) Inflammatoryand Bullous Skin Bowel Syndrome Desmoglein 2 Q14126|DSG2_HUMANDesmoglein-2 (SEQ ID NO: 505) Keratosis Pilaris Retinol Dehydrogenase 10Oily Skin Retinol Dehydrogenase 10 Osteoarthritis Hyaluronan SynthasePemphigus Vulgaris Plakophilin-1 Q13835|PKP1_HUMAN Plakophilin-1(isoform 2) (SEQ ID NO: 506) Pseudoxanthoma elasticum Elastinsp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) PsoriasisRetinol Dehydrogenase 10 Scar Treatment Tyrosinase P14679|TYRO_HUMANTyrosinase (isoform 1) (SEQ ID NO: 491) Scarring Elastinsp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) ScarringCollagen Type I P02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO:487) P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488)Scarring Collagen Type III P02461|CO3A1_HUMAN Collagen alpha-1(III)chain (isoform 1) (SEQ ID NO: 489) Skin Cancer Interferon Interferon,Alpha 1 P01562|IFNA1_HUMAN Interferon alpha-1/13 (SEQ ID NO: 530)Interferon, Alpha 2 P01563|IFNA2_HUMAN Interferon alpha-2 (SEQ ID NO:531) Interferon, Alpha 4 P05014|IFNA4_HUMAN Interferon alpha-4 (SEQ IDNO: 532) Interferon, Alpha 5 P01569|IFNA5_HUMAN Interferon alpha-5 (SEQID NO: 533) Interferon, Alpha 6 P05013|IFNA6_HUMAN Interferon alpha-6(SEQ ID NO: 534) Interferon, Alpha 7 P01567|IFNA7_HUMAN Interferonalpha-7 (SEQ ID NO: 535) Interferon, Alpha 8 P32881|IFNA8_HUMANInterferon alpha-8 (SEQ ID NO: 536) Interferon, Alpha 10P01566|IFN10_HUMAN Interferon alpha-10 (SEQ ID NO: 537) Interferon,Alpha 14 P01570|IFN14_HUMAN Interferon alpha-14 OS (SEQ ID NO: 538)Interferon, Alpha 16 P05015|IFN16_HUMAN Interferon alpha-16 (SEQ ID NO:539) Interferon, Alpha 17 P01571|IFN17_HUMAN Interferon alpha-17 (SEQ IDNO: 540) Interferon, Alpha 21 P01568|IFN21_HUMAN Interferon alpha-21(SEQ ID NO: 541) Interferon, Gamma P01579|IFNG_HUMAN Interferon gamma(SEQ ID NO: 542) Interferon, Beta P01574|IFNB_HUMAN Interferon beta (SEQID NO: 543) Interferon, Kappa Q9P0W0|IFNK_HUMAN Interferon kappa (SEQ IDNO: 544) Interferon, Epsilon Q86WN2|IFNE_HUMAN Interferon epsilon (SEQID NO: 545) Striate Palmoplantar Keratoderma ADAM17 P78536|ADA17_HUMANDisintegrin and metalloproteinase domain-containing protein 17 (isoformA) (SEQ ID NO: 502) Tanning Tyrosinase P14679|TYRO_HUMAN Tyrosinase(isoform 1) (SEQ ID NO: 491) Vitiligo Melanocyte-Stimulating HormoneAlpha-MSH P01189|138-150 (SEQ ID NO: 526) Beta-MSH P01189|217-234 (SEQID NO: 527) Gamma-MSH P01189|77-87 (SEQ ID NO: 528) ProopiomelanocortinP01189|COLI_HUMAN Pro-opiomelanocortin (SEQ ID NO: 529) VitiligoTyrosinase P14679|TYRO_HUMAN Tyrosinase (isoform 1) (SEQ ID NO: 491)Warts Interferon Interferon, Alpha 1 P01562|IFNA1_HUMAN Interferonalpha-1/13 (SEQ ID NO: 530) Interferon, Alpha 2 P01563|IFNA2_HUMANInterferon alpha-2 (SEQ ID NO: 531) Interferon, Alpha 4P05014|IFNA4_HUMAN Interferon alpha-4 (SEQ ID NO: 532) Interferon, Alpha5 P01569|IFNA5_HUMAN Interferon alpha-5 (SEQ ID NO: 533) Interferon,Alpha 6 P05013|IFNA6_HUMAN Interferon alpha-6 (SEQ ID NO: 534)Interferon, Alpha 7 P01567|IFNA7_HUMAN Interferon alpha-7 (SEQ ID NO:535) Interferon, Alpha 8 P32881|IFNA8_HUMAN Interferon alpha-8 (SEQ IDNO: 536) Interferon, Alpha 10 P01566|IFN10_HUMAN Interferon alpha-10(SEQ ID NO: 537) Interferon, Alpha 14 P01570|IFN14_HUMAN Interferonalpha-14 OS (SEQ ID NO: 538) Interferon, Alpha 16 P05015|IFN16_HUMANInterferon alpha-16 (SEQ ID NO: 539) Interferon, Alpha 17P01571|IFN17_HUMAN Interferon alpha-17 (SEQ ID NO: 540) Interferon,Alpha 21 P01568|IFN21_HUMAN Interferon alpha-21 (SEQ ID NO: 541)Interferon, Gamma P01579|IFNG_HUMAN Interferon gamma (SEQ ID NO: 542)Interferon, Beta P01574|IFNB_HUMAN Interferon beta (SEQ ID NO: 543)Interferon, Kappa Q9P0W0|IFNK_HUMAN Interferon kappa (SEQ ID NO: 544)Interferon, Epsilon Q86WN2|IFNE_HUMAN Interferon epsilon (SEQ ID NO:545) Wound Healing Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQID NO: 486) Wound Healing Collagen Type I P02452|CO1A1_HUMAN Collagenalpha-1(I) chain (SEQ ID NO: 487) P08123|CO1A2_HUMAN Collagen alpha-2(I)chain (SEQ ID NO: 488) Wound Healing Collagen Type IIIP02461|CO3A1_HUMAN Collagen alpha-1(III) chain (isoform 1) (SEQ ID NO:489) Xeroderma Pigmentosum DNA Polymerase Eta Q9Y253|POLH_HUMAN DNApolymerase eta (isoform 1) (SEQ ID NO: 507)

Additional illustrative targets of the present invention include thecosmetic targets listed in Table 6 of International Patent PublicationNo. WO 2013/151671, the contents of which are hereby incorporated byreference in their entirety.

In various embodiments, the agents of the present invention are used inmethods the effect the integumentary system of a human. The presentcompositions and methods may be used to alter a biological and/orphysiological process to, for example, reduce skin sagging, increaseskin thickness, increase skin volume, reduce the number of wrinkles, thelength of wrinkles and/or the depth of wrinkles, increase skintightness, firmness, tone and/or elasticity, increase skin hydration andability to retain moisture, water flow and osmotic balance, increase thelevels of skin lipids; increase the extracellular matrix and/or adhesionand communication polypeptides; increase skin energy production;utilization and conservation; improve oxygen utilization; improve skincell life; improve skin cell immunity defense, heat shock stressresponse, antioxidant defense capacity to neutralize free radicals,and/or toxic defense; improve the protection and recovery fromultraviolet rays; improve skin cell communication and skin cellinnervations; improve cell cohesion/adhesion; improve calcium mineraland other mineral metabolism; improve cell turnover; and improve cellcircadian rhythms.

Further still, in some embodiments, the present compositions may be usedin the treatment, control, or prevention of a disease, disorder and/orcondition and/or may alter, modify or change the appearance of a memberof the integumentary system of a subject suffering from a disease,disorder and/or condition such as, but not limited to, acne vulgaris,acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acnekeloidalis nuchae, acne mechanica, acne medicamentosa, acne miliarisnecrotica, acne necrotica, acne rosacea, actinic keratosis, acnevulgaris, acne aestivalis, acne conglobata, acne cosmetic, acnefulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa,acne miliaris necrotica, acne necrotica, acne rosacea, acute urticaria,allergic contact dermatitis, alopecia areata, angioedema, athlete'sfoot, atopic dermatitis, autoeczematization, baby acne, balding,bastomycosis, blackheads, birthmarks and other skin pigmentationproblems, boils, bruises, bug bites and stings, burns, cellulitis,chiggers, chloracne, cholinergic or stress uricara, chronic urticara,cold type urticara, confluent and reticulated papillomatosis, corns,cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidroticeczema, diaper rash, dry skin, dyshidrosis, ectodermal dysplasia suchas, hyprohidrotic ectodermal dysplasia and X-linked hyprohidroticectodermal dysplasia, eczema, epidermaodysplasia verruciformis, erythemanodosum, excoriated acne, exercise-induced anaphylasis folliculitis,excess skin oil, folliculitis, freckles, frostbite, fungal nails, hairdensity, hair growth rate, halogen acne, hair loss, heat rash, hematoma,herpes simplex infections (e.g. non-genital), hidradenitis suppurativa,hives, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermaldysplasia, hypopigmentation, impetigo, ingrown hair, heat type urticara,ingrown toenail, infantile acne or neonatal acne, itch, irritant contactdermatitis, jock itch, keloid, keratosis pilaris, lichen planus, lichensclerosus, lupus miliaris disseminatus faciei, melasma, moles, molluscumcontagiosum, nail growth rate, nail health, neurodermatitis, nummulareczema, occupational acne, oil acne, onychomycosis, physical urticara,pilonidal cyst, pityriasis rosea, pityriasis versicolor, poison ivy,pomade acne, pseudofolliculitis barbae or acne keloidalis nuchae,psoriasis, psoriatic arthritis, pressure or delayed pressue urticara,puncture wounds such as cuts and scrapes, rash, rare or water typeurticara, rhinoplasty, ringworm, rosacea, rothmund-thomson syndrome,sagging of the skin, scabis, scars, seborrhea, seborrheic dermatitis,shingles, skin cancer, skin tag, solar type urticara, spider bite,stretch marks, sunburn, tar acne, tropical acne, thinning of skin,thrush, tinea versicolor, transient acantholytic dermatosis, tycoon'scap or acne necrotica miliaris, uneven skin tone, varicose veins, venouseczema, vibratory angioedema, vitiligo, warts, Weber-Christian disease,wrinkles, x-linked hypohidrotic ectodermal dysplasia, xerotic eczema,yeast infection and general signs of aging.

In some embodiments, there is provided methods of treating, controllingor preventing dry skin with the present compositions. In someembodiments profilaggrin (a protein which is converted to filaggrin) isa protein of interest (e.g. when treating ichthyosis vulgaris).

In some embodiments, there is provided methods of treating, controllingor preventing any one of the various types of psoriasis (e.g. plaguepsoriasis, guttate psoriasis, pustular psoriasis, inverse psoriasis, anderythrodermic psoriasis). In various embodiments, the protein ofinterest is any of the products of the genes psoriasis susceptibility 1through 9 (PSORSI-PSORS9).

Various embodiments relate to the treatment, control, or prevention ofeczema (e.g. atopic dermatitis, nummular eczema, dyshidrotic eczema,seborrheic dermatitis, irritant contact dermatitis, allergic contactdermatitis, dyshidrosis, venous eczema, dermatitis herpetiformis,neurodermatitis, autoeczematization and xerotic eczema) and, optionally,one or more of the following may be targeted: filaggrin; three geneticvariants, ovo-like 1 (OVOL1), actin-like 9 (ACTL9) and kinesin familymember 3 A (KIF3A) have been associated with eczema; and the genesbrain-derived neurotrophic factor (BDNF) and tachykinin, precursor 1(TAC1).

Hives, or urticaria, including, but not limited to, acute urticaria,chronic urticara and angioedema, physical urticara, pressure or delayedpressue urticara, cholinergic or stress uricara, cold type urticara,heat type urticara, solar type urticara, rare or water type urticara,vibratory angioedema, exercise-induced anaphylasis and dermatographismmay be treated with the present compositions by, for example, targetingPLCG-2.

Various embodiments relate to the treatment, control, or prevention ofrosacea, which includes, but is not limited to, erthematotelangiectaticrosacea, papulopustular rosacea, phymatous rosacea, and ocular rosacea.Optionally, cathelicidin antimicrobial peptide (CAMP) and/orkallikrein-related peptidase 5 (also known as stratum corneum trypticenzyme (SCTE)) are proteins of interest.

In some embodiments, there is provided methods of treating, controllingor preventing acne with the present compositions. For example, acne mayinclude, but is not limited to, acneiform eruptions, acne aestivalis,acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae,acne mechanica, acne medicamentosa, acne miliaris necrotica, acnenecrotica, acne rosacea, baby acne, blackheads, chloracne, excoriatedacne, halogen acne, infantile acne or neonatal acne, lupus miliarisdisseminatus faciei, occupational acne, oil acne, pomade acne, tar acne,tropical acne, tycoon's cap or acne necrotica miliaris,pseudofolliculitis barbae or acne keloidalis nuchae, and hidradenitissuppurativa. In these embodiments, the protein of interest may be one ormore matrix metalloproteinases (MMP), e.g., matrix metalloproteinase-1(MMP-1 or interstitial collagenase), matrix metalloproteinase-9 (MMP-9),and matrix metalloproteinase-13 (MMP-13).

In further embodiments, vitiligo is treated with the presentcompositions, e.g. wherein the NLR family, pyrin domain containing 1gene (NALP1) gene is targeted.

In some embodiments, the present compositions find use in the treatment,control, or prevention of hyprohidrotic ectodermal dysplasia (HED), e.g.via the ectodysplasin A gene (EDA), receptor (EDAR), and receptorassociated death domain (EDARADD).

In some embodiments, the present compositions find use in the treatment,control, or prevention of balding, or hair thinning (e.g. male patternbaldness, or androgenetic alopecia (AGA)) and, optionally, one or moreof the following may be the protein of interest: androgen receptor (AR),ectodysplasin A2 receptor (EDA2R) and lysophosphatidic acid receptor 6(P2RY5).

The present compositions also find use in methods of treatment, control,or prevention of scars and stretch marks (striae), e.g. via collagen,ribosomal s6 kinase, sectrected phosphoprotein 1 (also known asosteopontin), or transforming growth factor beta 3.

Epidermodysplasia verruciformis (also known as Lutz-Lewandowskyepidermodysplasia), a rare autosomal recessive genetic hereditary skindisorder, may also be treated with compositions of the presentinvention, e.g. by targeted transmembrane channel-like 6 (EVER1) ortransmembrane channellike 8 (EVER2) genes.

In some embodiments, skin sagging, thinning or wrinkling may be treated,controlled or prevented with present composition, e.g. by targeting oneor more of the proteins of interest such as collagen, elastin,fibroblast growth factor 7, TIMP metallopeptidase inhibitors, matrixmetallopeptidases, superoxide dismutase and other extracellular matrixproteins and proteoglycans.

Further embodiments are used in tanning of the skin, such as viamelanocyte-stimulating hormone and/or proopiomelanocortin.

In some embodiments, the present compositions may be used for woundtreatment. In some embodiments, methods of treating, controlling orpreventing wounds with the present compositions comprises additionalsteps of, for example, cleaning the wound bed to facilitate woundhealing and closure, including, but not limited to: debridement, sharpdebridement (surgical removal of dead or infected tissue from a wound),optionally including chemical debriding agents, such as enzymes, toremove necrotic tissue; wound dressings to provide the wound with amoist, warm environment and to promote tissue repair and healing (e.g.,wound dressings comprising hydrogels (e.g., AQUASORB; DUODERM),hydrocolloids (e.g., AQUACEL; COMFEEL), foams (e.g., LYOFOAM;SPYROSORB), and alginates (e.g., ALGISITE; CURASORB); administration ofgrowth factors to stimulate cell division and proliferation and topromote wound healing e.g. becaplermin; and (iv) soft-tissue woundcoverage, a skin graft may be necessary to obtain coverage of clean,non-healing wounds (e.g., autologous skin grafts, cadaveric skin graft,bioengineered skin substitutes (e.g., APLIGRAF; DERMAGRAFT)).

In various embodiments, the nucleic acid drug described herein can beused in a variety of cosmetic/plastic surgery procedures, including,without limitation, a surgical procedure involving skin grafting and anaesthetic or cosmetic surgery (e.g. a facial plastic surgery procedureincluding, but not limited to blepharoplasty, rhinoplasty, rhytidectomy,genioplasty, facial implants, otoplasty, hair implantation, cleft lipand cleft palate repair, and/or a body plastic surgery procedureincluding but not limited to abdominoplasty, brachioplasty, thigh lift,breast reduction, breast augmentation, body contouring, liposuction,hand surgery).

In various embodiments, a variety of cancers are treated, controlled orprevented with the present compositions (e.g., colorectal cancer,gallbladder cancer, lung cancer, pancreatic cancer, and stomach cancer).In some embodiments, skin cancer is treated with the presentcompositions. For instance, the skin cancer is one or more of actinickeratosis, basal cell carcinoma, melanoma, Kaposi's sarcoma, andsquamous cell carcinoma. In some embodiments, the present compositionsare used adjuvant to complete circumferential peripheral and deep marginassessment, Mohs surgery, radiation (e.g. external beam radiotherapy orbrachytherapy), chemotherapy (including but not limited to topicalchemotherapy, e.g. with imiquimod or 5-fluorouracil), and cryotherapy.The present compositions also find use in the treatment of variousstages of cancers, including skin cancers (e.g. basal cell cancer (BCC),squamous cell cancer (SCC), and melanoma), such as a stage of theAmerican Joint Committee on Cancer (AJCC) TNM system (e.g. one or moreof TX, T0, Tis, T1, T1a, T1b, T2, T2A, T2B, T3, T3a, T3b, T4, T4a, T4b,NX, N0, N1, N2, N3, M0, M1a, M1b, M1c) and/or a staging system (e.g.Stage 0, Stage IA, Stage IB, Stage IIA, Stage IIB, Stage IIC, StageIIIA, Stage IIIB, Stage IIIC, Stage IV).

Illustrative cancers and/or tumors of the present invention include, butare not limited to, a basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and central nervous system cancer;breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

In various embodiments, one or more rare diseases are treated,controlled or prevented with the present compositions, including, by wayof illustration, Erythropoietic Protoporphyria, Hailey-Hailey Disease,Epidermolysis Bullosa (EB), Xeroderma Pigmentosum, Ehlers-DanlosSyndrome, Cutis Laxa, Protein C & Protein S Deficiency, Alport Syndrome,Striate Palmoplantar Keratoderma, Lethal Acantholytic EB, PseudoxanthomaElasticum (PXE), Ichthyosis Vulgaris, Pemphigus Vulgaris, and Basal CellNevus Syndrome.

In various embodiments, the present compositions are used to treat,control or prevent one or more inflammatory diseases or conditions, suchas inflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopicdermatitis, septic shock, rheumatoid arthritis, inflammatory boweldisease, inflammatory pelvic disease, pain, ocular inflammatory disease,celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familialeosinophilia (FE), autosomal recessive spastic ataxia, laryngealinflammatory disease; Tuberculosis, Chronic cholecystitis,Bronchiectasis, Silicosis and other pneumoconioses.

In various embodiments, the present compositions are used to treat,control or prevent one or more autoimmune diseases or conditions, suchas multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn'sdisease, ulcerative colitis, Guillain-Barre syndrome, scleroderms,Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy,Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosingcholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto'sthyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection(e.g., prevention of allograft rejection) pernicious anemia, rheumatoidarthritis, systemic lupus erythematosus, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In various embodiments, the present compositions are used to treat,control or prevent one or more neurologic diseases, including ADHD,AIDS—Neurological Complications, Absence of the Septum Pellucidum,Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis,Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, AicardiSyndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia,Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly,Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia,Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation,Arteriovenous Malformation, Aspartame, Asperger Syndrome, AtaxiaTelangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder,Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, BattenDisease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm,Benign Focal Amyotrophy, Benign Intracranial Hypertension,Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm,Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, BrachialPlexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, BrainInjury, Brain and Spinal Tumors, Brown-Sequard Syndrome, BulbospinalMuscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia,Cavernomas, Cavernous Angioma, Cavernous Malformation, Central CervicalCord Syndrome, Central Cord Syndrome, Central Pain Syndrome, CephalicDisorders, Cerebellar Degeneration, Cerebellar Hypoplasia, CerebralAneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, CerebralBeriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy,Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder,Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance,Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma,including Persistent Vegetative State, Complex Regional Pain Syndrome,Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy,Congenital Vascular Cavernous Malformations, Corticobasal Degeneration,Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease,Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic InclusionBody Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-DancingFeet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier'sSyndrome, Dejerine-Klumpke Palsy, Dementia—Multi-Infarct,Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis,Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, DiffuseSclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia,Dysphagia, Dyspraxia, Dystonias, Early Infantile EpilepticEncephalopathy, Empty Sella Syndrome, Encephalitis Lethargica,Encephalitis and Meningitis, Encephaloceles, Encephalopathy,Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenneand Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome,Fainting, Familial Dysautonomia, Familial Hemangioma, FamilialIdiopathic Basal Ganglia Calcification, Familial Spastic Paralysis,Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, FloppyInfant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann'sSyndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis,Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy,Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 AssociatedMyelopathy, Hallervorden-Spatz Disease, Head Injury, Headache,Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, HereditaryNeuropathies, Hereditary Spastic Paraplegia, Heredopathia AtacticaPolyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, HirayamaSyndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly,Hydrocephalus—Normal Pressure, Hydrocephalus, Hydromyelia,Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia,Immune-Mediated Encephalomyelitis, Inclusion Body Myositis,Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic AcidStorage Disease, Infantile Refsum Disease, Infantile Spasms,Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts,Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome,Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Klüver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenicSyndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-InSyndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, LymeDisease—Neurological Complications, Machado-Joseph Disease,Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome,Meningitis, Menkes Disease, Meralgia Paresthetica, MetachromaticLeukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome,Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, MonomelicAmyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses,Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal MotorNeuropathy, Multiple Sclerosis, Multiple System Atrophy with OrthostaticHypotension, Multiple System Atrophy, Muscular Dystrophy,Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic DiffuseSclerosis, Myoclonic Encephalopathy of Infants, Myoclonus,Myopathy—Congenital, Myopathy—Thyrotoxic, Myopathy, Myotonia Congenita,Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with BrainIron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,Neurological Complications of AIDS, Neurological Manifestations of PompeDisease, Neuromyelitis Optica, Neuromyotonia, Neuronal CeroidLipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary,Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy,Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome,Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson'sDisease, Parnyotonia Congenita, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir IISyndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy,Periventricular Leukomalacia, Persistent Vegetative State, PervasiveDevelopmental Disorders, Phytanic Acid Storage Disease, Pick's Disease,Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease,Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia,Postinfectious Encephalomyelitis, Postural Hypotension, PosturalOrthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, PrimaryLateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy,Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent andPyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I,Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and otherautoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, RefsumDisease-Infantile, Refsum Disease, Repetitive Motion Disorders,Repetitive Stress Injuries, Restless Legs Syndrome,Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome,Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint VitusDance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease,Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, SevereMyoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles,Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness,Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction,Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy,Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome,Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-WeberSyndrome, Subacute Sclerosing Panencephalitis, SubcorticalArteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea,Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia,Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, TarlovCysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal CordSyndrome, Thomsen Disease, Thoracic Outlet Syndrome, ThyrotoxicMyopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, TransientIschemic Attack, Transmissible Spongiform Encephalopathies, TransverseMyelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, TropicalSpastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor,Vasculitis including Temporal Arteritis, Von Economo's Disease, VonHippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, WestSyndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease,X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

In various embodiments, the present compositions are used to treat oneor more respiratory diseases, such as asthma, chronic obstructivepulmonary disease (COPD), bronchiectasis, allergic rhinitis, sinusitis,pulmonary vasoconstriction, inflammation, allergies, impededrespiration, respiratory distress syndrome, cystic fibrosis, pulmonaryhypertension, pulmonary vasoconstriction, emphysema, Hantaviruspulmonary syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome,Pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, Sarcoidosis,complications associated with respiratory syncitial virus infection, andother respiratory diseases.

In various embodiments, the present compositions are used to treat,control or prevent cardiovascular disease, such as a disease orcondition affecting the heart and vasculature, including but not limitedto, coronary heart disease (CHD), cerebrovascular disease (CVD), aorticstenosis, peripheral vascular disease, atherosclerosis,arteriosclerosis, myocardial infarction (heart attack), cerebrovasculardiseases (stroke), transient ischaemic attacks (TIA), angina (stable andunstable), atrial fibrillation, arrhythmia, vavular disease, and/orcongestive heart failure.

In various embodiments, the present compositions are used to treat,control or prevent one or more metabolic-related disorders. In variousembodiments, the present invention is useful for the treatment,controlling or prevention of diabetes, including Type 1 and Type 2diabetes and diabetes associated with obesity. The compositions andmethods of the present invention are useful for the treatment orprevention of diabetes-related disorders, including without limitationdiabetic nephropathy, hyperglycemia, impaired glucose tolerance, insulinresistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia,hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDLlevels, atherosclerosis and its sequelae, vascular restenosis, irritablebowel syndrome, inflammatory bowel disease, including Crohn's diseaseand ulcerative colitis, other inflammatory conditions, pancreatitis,abdominal obesity, neurodegenerative disease, retinopathy, neoplasticconditions, adipose cell tumors, adipose cell carcinomas, such asliposarcoma, prostate cancer and other cancers, including gastric,breast, bladder and colon cancers, angiogenesis, Alzheimer's disease,psoriasis, high blood pressure, Metabolic Syndrome (e.g. a person hasthree or more of the following disorders: abdominal obesity,hypertriglyceridemia, low HDL cholesterol, high blood pressure, and highfasting plasma glucose), ovarian hyperandrogenism (polycystic ovarysyndrome), and other disorders where insulin resistance is a component,such as sleep apnea. The compositions and methods of the presentinvention are useful for the treatment, control, or prevention ofobesity, including genetic or environmental, and obesity-relateddisorders. The obesity-related disorders herein are associated with,caused by, or result from obesity. Examples of obesity-related disordersinclude obesity, diabetes, overeating, binge eating, and bulimia,hypertension, elevated plasma insulin concentrations and insulinresistance, dyslipidemia, hyperlipidemia, endometrial, breast, prostate,kidney and colon cancer, osteoarthritis, obstructive sleep apnea,gallstones, heart disease, abnormal heart rhythms and arrythmias,myocardial infarction, congestive heart failure, coronary heart disease,sudden death, stroke, polycystic ovary disease, craniopharyngioma,Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects,normal variant short stature, Turner's syndrome, and other pathologicalconditions showing reduced metabolic activity or a decrease in restingenergy expenditure as a percentage of total fat-free mass, e.g, childrenwith acute lymphoblastic leukemia. Further examples of obesity-relateddisorders are Metabolic Syndrome, insulin resistance syndrome,reproductive hormone abnormalities, sexual and reproductive dysfunction,such as impaired fertility, infertility, hypogonadism in males andhirsutism in females, fetal defects associated with maternal obesity,gastrointestinal motility disorders, such as obesity-relatedgastro-esophageal reflux, respiratory disorders, such asobesity-hypoventilation syndrome (Pickwickian syndrome), breathlessness,cardiovascular disorders, inflammation, such as systemic inflammation ofthe vasculature, arteriosclerosis, hypercholesterolemia, lower backpain, gallbladder disease, hyperuricemia, gout, and kidney cancer, andincreased anesthetic risk. The compositions and methods of the presentinvention are also useful to treat Alzheimer's disease.

Nucleic acids, including liposomal formulations containing nucleicacids, when delivered in vivo, can accumulate in the liver and/orspleen. It has now been discovered that nucleic acids encoding proteinscan modulate protein expression in the liver and spleen, and thatnucleic acids used in this manner can constitute potent therapeutics forthe treatment of liver and spleen diseases. Certain embodiments aretherefore directed to a method for treating liver and/or spleen diseaseby delivering to a patient a nucleic acid encoding a protein ofinterest. Other embodiments are directed to a therapeutic compositioncomprising a nucleic acid encoding a protein of interest, for thetreatment of liver and/or spleen disease. Diseases and conditions of theliver and/or spleen that can be treated include, but are not limited to:hepatitis, alcohol-induced liver disease, drug-induced liver disease,Epstein Barr virus infection, adenovirus infection, cytomegalovirusinfection, toxoplasmosis, Rocky Mountain spotted fever, non-alcoholicfatty liver disease, hemochromatosis, Wilson's Disease, Gilbert'sDisease, and cancer of the liver and/or spleen.

In some embodiments, the present compositions and methods relate to thetreatment of type 1 diabetes, heart disease, including ischemic anddilated cardiomyopathy, macular degeneration, Parkinson's disease,cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severecombined immunodeficiency, hereditary sensory neuropathy, xerodermapigmentosum, Huntington's disease, muscular dystrophy, amyotrophiclateral sclerosis, Alzheimer's disease, cancer, and infectious diseasesincluding hepatitis and HIV/AIDS.

In various embodiments, the present methods and compositions find use intreating or preventing one or more metabolic diseases or disorders. Invarious embodiments, the present methods and compositions find use intreating or preventing one or more of diseases or disorders ofcarbohydrate metabolism. diseases or disorders of amino acid metabolism,diseases or disorders of the urea cycle, diseases or disorders of fattyacid metabolism, diseases or disorders of porphyrin metabolism,lysosomal storage disorders, peroxisome biogenesis disorders, anddiseases or disorders of purine or pyrimidine metabolism.

In various embodiments, the present methods and compositions find use intreating or preventing one or more of diseases or disorders in the tablebelow. In various embodiments, the present methods and compositions finduse in treating or preventing one or more of diseases or disorders inthe table below for instance by modulating the genes associated with thediseases in the table below. In some embodiments, the present methodsand compositions find use in gene-editing the genes described in thetable below using the present compositions.

Category Disease Genes Entrez ID Disorders of Galactosemia GALT, GALK1,GALE 2592, 2584, 2582 carbohydrate Essential fructosuria KHK 3795metabolism Hereditary fructose ALDOB 229 intolerance Glycogen storagedisease G6PC, SLC37A4, SLC17A3 2538, 2542, 10786 type I Glycogen storagedisease GAA 2548 type II Glycogen storage disease AGL 178 type IIIGlycogen storage disease GBE1 2632 type IV Glycogen storage disease PYGM5837 type V Glycogen storage disease PYGL 5836 type VI Glycogen storagedisease PYGM 5837 type VII Glycogen storage disease PHKA1, PHKA2, PHKB,5255, 5256, 5257, 5260, type IX PHKG1, PHKG2 5261 Glycogen storagedisease SLC2A2 6514 type XI Glycogen storage disease ALDOA 226 type XIIGlycogen storage disease ENO1, ENO2, ENO3 2023, 2026, 2027 type XIIIGlycogen storage disease GYS1, GYS2 2997, 2998 type 0 Pyruvatecarboxylase PC 5091 deficiency Pyruvate kinase deficiency PKLR 5313Transaldolase deficiency TALDO1 6888 Triosephosphate isomerase TPI1 7167deficiency Fructose bisphosphatase FBP1 2203 deficiency HyperoxaluriaAGXT, GRHPR  189, 9380 Hexokinase deficiency HK1 3098 Glucose-galactoseSLC5A1 6523 malabsorption Glucose-6-phosphate G6PD 2539 dehydrogenasedeficiency Disorders of Alkaptonuria HGD 3081 amino acidAspartylglucosaminuria AGA 175 metabolism Methylmalonic acidemia MUT,MCEE, MMAA, 4594, 84693, 166785, MMAB, MMACHC, 326625, 25974, 27249,MMADHC, LMBRD1 55788 Maple syrup urine disease BCKDHA, BCKDHB, DBT, 593,594, 1629, 1738 DLD Homocystinuria CBS 875 Tyrosinemia FAH, TAT, HPD2184, 6898, 3242 Trimethylaminuria FMO3 2328 Hartnup disease SLC6A19340024 Biotinidase deficiency BTD 686 Ornithine OTC 5009carbamoyltransferase deficiency Carbamoyl-phosphate CPS1 1373 synthase Ideficiency disease Citrullinemia ASS, SLC25A13  445, 10165Hyperargininemia ARG1 383 Hyperhomocysteinemia MTHFR 4524Hypermethioninemia MAT1A, GNMT, AHCY 4143, 27232, 191 HyperlysinemiasAASS 10157 Nonketotic hyperglycinemia GLDC, AMT, GCSH 2731, 275, 2653Propionic acidemia PCCA, PCCB 5095, 5096 Hyperprolinemia ALDH4A1, PRODH8659, 5625 Cystinuria SLC3A1, SLC7A9  6519, 11136 Dicarboxylicaminoaciduria SLC1A1 6505 Glutaric acidemia type 2 ETFA, ETFB, ETFDH2108, 2109, 2110 Isovaleric acidemia IVD 3712 2-Hydroxyglutaric aciduriaL2HGDH, D2HGDH  79944, 728294 Disorders of the N-Acetylglutamate NAGS162417 urea cycle synthase deficiency Argininosuccinic aciduria ASL 435Argininemia ARG1 383 Disorders of Very long-chain acyl- ACADVL 37 fattyacid coenzyme A metabolism dehydrogenase deficiency Long-chain3-hydroxyacyl- HADHA 3030 coenzyme A dehydrogenase deficiencyMedium-chain acyl- ACADM 34 coenzyme A dehydrogenase deficiencyShort-chain acyl-coenzyme ACADS 35 A dehydrogenase deficiency3-hydroxyacyl-coenzyme A HADH 3033 dehydrogenase deficiency 2,4Dienoyl-CoA reductase NADK2 133686 deficiency3-Hydroxy-3-methylglutaryl- HMGCL 3155 CoA lyase deficiency Malonyl-CoAMLYCD 23417 decarboxylase deficiency Systemic primary carnitine SLC22A56584 deficiency Carnitine-acylcarnitine SLC25A20 788 translocasedeficiency Carnitine CPT1A 1374 palmitoyltransferase I deficiencyCarnitine CPT2 1376 palmitoyltransferase II deficiency Lysosomal acidlipase LIPA 3988 deficiency Gaucher's disease GBA 2629 Disorders ofAcute intermittent porphyria HMBS 3145 porphyrin Gunther disease UROS7390 metabolism Porphyria cutanea tarda UROD 7389 HepatoerythropoieticUROD 7389 porphyria Hereditary coproporphyria CPOX 1371 Variegateporphyria PPOX 5498 Erythropoietic FECH 2235 protoporphyriaAminolevulinic acid ALAD 210 dehydratase deficiency porphyria LysosomalFarber disease ASAH1 427 storage Krabbe disease GALC 2581 disordersGalactosialidosis CTSA 5476 Fabry disease GLA 2717 Schindler diseaseNAGA 4668 GM1 gangliosidosis GLB1 2720 Tay-Sachs disease HEXA 3073Sandhoff disease HEXB 3074 GM2-gangliosidosis, AB GM2A 2760 variantNiemann-Pick disease SMPD1, NPC1, NPC2 6609, 4864, 10577 MetachromaticARSA, PSAP  410, 5660 leukodystrophy Multiple sulfatase SUMF1 285362deficiency Hurler syndrome IDUA 3425 Hunter syndrome IDS 3423 Sanfilipposyndrome SGSH, NAGLU, HGSNAT, 6448, 4669, 138050, 2799 GNS Morquiosyndrome GALNS, GLB1 2588, 2720 Maroteaux-Lamy syndrome ARSB 411 Slysyndrome GUSB 2990 Sialidosis NEU1, NEU2, NEU3, NEU4 4758, 4759, 10825,129807 I-cell disease GNPTAB, GNPTG 79158, 84572 Mucolipidosis type IVMCOLN1 57192 Infantile neuronal ceroid PPT1, PPT2 5538, 9374lipofuscinosis Jansky-Bielschowsky TPP1 1200 disease Batten diseaseCLN1, CLN2, CLN3, CLN5, 5538, 1200, 1201, 1203, CLN6, MFSD8, CLN8, CTSD54982, 256471, 2055, 1509 Kufs disease, Type A CLN6, PPT1 54982, 5538 Kufs disease, Type B DNAJC5, CTSF 80331, 8722  Alpha-mannosidosisMAN2B1, MAN2B2, 4125, 23324, 4123 MAN2C1 Beta-mannosidosis MANBA 4226Fucosidosis FUCA1 2517 Cystinosis CTNS 1497 Pycnodysostosis CTSK 1513Salla disease SLC17A5 26503 Infantile free sialic acid SLC17A5 26503storage disease Danon disease LAMP2 3920 Peroxisome Zellweger syndromePEX1, PEX2, PEX3, PEX5 5189, 5828, 8504, 5830, biogenesis PEX6, PEX12,PEX14, 5190, 5193, 5195, 55670 disorders PEX26 Infantile Refsum diseasePEX1, PEX2, PEX26 5189, 5828, 55670 Neonatal PEX5, PEX1, PEX10, 5830,5189, 5192, 5194, adrenoleukodystrophy PEX13, PEX26 55670 RCDP Type 1PEX7 5191 Pipecolic acidemia PAHX 5264 Acatalasia CAT 847 Hyperoxaluriatype 1 AGXT 189 Acyl-CoA oxidase ACOX1 51 deficiency D-bifunctionalprotein HSD17B4 3295 deficiency Dihydroxyacetonephosphate GNPAT 8443acyltransferase deficiency X-linked ABCD1 215 adrenoleukodystrophyα-Methylacyl-CoA AMACR 23600 racemase deficiency RCDP Type 2 DHAPAT 8443RCDP Type 3 AGPS 8540 Adult Refsum disease-1 PHYH 5264 Mulibrey nanismTRIM37 4591 Disorders of Lesch-Nyhan syndrome HPRT 3251 purine orAdenine APRT 353 pyrimidine phosphoribosyltransferase metabolismdeficiency Adenosine deaminase ADA 100 deficiency Adenosinemonophosphate AMPD1 270 deaminase deficiency type 1 Adenylosuccinatelyase ADSL 158 deficiency Dihydropyrimidine DPYD 1806 dehydrogenasedeficiency Miller syndrome DHODH 1723 Orotic aciduria UMPS 7372 Purinenucleoside PNP 4860 phosphorylase deficiency Xanthinuria XDH, MOCS1,MOCS2, 7498, 4337, 4338, 10243 GEPH

The Entrez entries listed in the table above are hereby incorporated byreference in their entireties.

Further, in some embodiments, the present methods and compositions finduse in targeting any of the proteins or in treatment of any of thediseases or disorders of Table 2B. In various embodiments, the presentinvention contemplates the targeting of the full-length and/or truncatedforms of any of the proteins disclosed in Table 2B. In variousembodiments, the present invention contemplates the targeting of theprecursor forms and/or mature forms and/or isoforms of any of theproteins disclosed in Table 2B.

In various embodiments, the present invention contemplates the targetingof a protein having about 60% (e.g. about 60%, or about 61%, or about62%, or about 63%, or about 64%, or about 65%, or about 66%, or about67%, or about 68%, or about 69%, or about 70%, or about 71%, or about72%, or about 73%, or about 74%, or about 75%, or about 76%, or about77%, or about 78%, or about 79%, or about 80%, or about 81%, or about82%, or about 83%, or about 84%, or about 85%, or about 86%, or about87%, or about 88%, or about 89%, or about 90%, or about 91%, or about92%, or about 93%, or about 94%, or about 95%, or about 96%, or about97%, or about 98%, or about 99%) sequence identity with any of theprotein sequences disclosed herein (e.g. in Table 2B).

In various embodiments, the present invention contemplates the targetingof a protein comprising an amino acid sequence having one or more aminoacid mutations relative to any of the protein sequences described herein(e.g. in Table 2B). For example, the present invention contemplates thetargeting of a protein comprising an amino acid sequence having 1, or 2,or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12 amino acidmutations relative to any of the protein sequences described herein(e.g. in Table 2B). In some embodiments, the one or more amino acidmutations may be independently selected from substitutions, insertions,deletions, and truncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

In Table 2B, all Illustrative Identifiers (e.g. Gene Seq nos. andreferences are hereby incorporated by reference in their entireties).

TABLE 2B Illustrative Proteins, Illustrative Peptides, and IllustrativeIndications Protein/Peptide Illustrative Identifier ReferenceDescription/Illustrative Indication(s) Transthyretin (TTR) his geneencodes transthyretin, one of the three prealbumins including alpha-1-(SEQ ID NOs: 637 and antitrypsin, transthyretin and orosomucoid.Transthyretin is a carrier protein; it 638) transports thyroid hormonesin the plasma and cerebrospinal fluid, and also Gene ID: 7276 transportsretinol (vitamin A) in the plasma. The protein consists of a tetramer ofidentical subunits. More than 80 different mutations in this gene havebeen reported; most mutations are related to amyloid deposition,affecting predominantly peripheral nerve and/or the heart, and a smallportion of the gene mutations is non- amyloidogenic. The diseases causedby mutations include amyloidotic polyneuropathy, euthyroidhyperthyroxinaemia, amyloidotic vitreous opacities, cardiomyopathy,oculoleptomeningeal amyloidosis, meningocerebrovascular amyloidosis,carpal tunnel syndrome, etc. Endothelial Cell Specific This gene encodesa secreted protein which is mainly expressed in the endothelial Molecule1 cells in human lung and kidney tissues. The expression of this gene isregulated by (SEQ ID NO: 629 to 632) cytokines, suggesting that it mayplay a role in endothelium-dependent pathological Gene ID: 11082disorders. The transcript contains multiple polyadenylation and mRNAinstability signals. Two transcript variants encoding different isoformshave been found for this gene Parathyroid hormone PTH is secreted by thechief cells of the parathyroid glands. PTH plays an importantP01270|PTHY_HUMAN role in the regulation of serum calcium, serumphosphate, and vitamin D synthesis. Parathyroid hormone (SEQ ID NO: 508)BMP-1 GeneSeq BMP1 belongs to the transforming growth factor-beta (TGFB)super-family. Bone Accession P80618 morphogenic proteins inducecartilage and bone formation, play important role in WO8800205nephrogesis, and play an important role in the development of manyorgans, P13497/BMP1_HUMAN including lung, heart, teeth, gut, skin, andparticularly the kidney. BMP-1 activity can Bone morphogenetic bedetermined using the following assays known in the art: Nat Genet. 2001Jan; protein 1 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302;J Biol Chem, Vol. 274, (isoform BMP1-3) Issue 16, 10897-10902, Apr. 16,1999; and Hogan, B. L. M. (1996) Genes Dev. 10, (SEQ ID NO: 169)1580-1594. Induction of Cartilage, Tissue and Bone Growth, and DiabetesP13497-2|BMP1_HUMAN Isoform BMP1-1 of Bone morphogenetic protein 1(isoform BMP1-1) (SEQ ID NO: 509) P13497-3|BMP1_HUMAN Isoform BMP1-4 ofBone morphogenetic protein 1 (isoform BMP1-4) (SEQ ID NO: 510)P13497-4|BMP1_HUMAN Isoform BMP1-5 of Bone morphogenetic protein 1(isoform BMP1-5) (SEQ ID NO: 511) P13497-5|BMP1_HUMAN Isoform BMP1-6 ofBone morphogenetic protein 1 (isoform BMP1-6) (SEQ ID NO: 512)P13497-6|BMP1_HUMAN Isoform BMP1-7 of Bone morphogenetic protein 1(isoform BMP1-7) (SEQ ID NO: 513) BMP-2 GeneSeq BMP-2 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone AccessionP80619 morphogenic protein induces bone formation. BMP-2 activity can bedetermined WO8800205 using the following assays known in the art: NatGenet. 2001 Jan; 27(1): 84-8; Eur J P12643/BMP2_HUMAN Biochem 1996 Apr.1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bonemorphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10,1580-1594. protein 2 Induction of Cartilage, Tissue and Bone Growth, andDiabetes. Infarction recovery. (SEQ ID NO: 170) Bone repair.Osteoporosis. BMP-3 BMP-3 belongs to the transforming growth factor-beta(TGFB) superfamily. Bone P12645|BMP3_HUMAN morphogenic protein inducesbone formation. BMP-3 activity can be determined Bone morphogeneticusing the following assays known in the art: Nat Genet. 2001 Jan; 27(1):84-8; Eur J protein 3 Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem,Vol. 274, Issue 16, 10897-10902, (SEQ ID NO: 514) Apr. 16, 1999; andHogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage,Tissue and Bone Growth, and Diabetes BMP-2B GeneSeq BMP-2b belongs tothe transforming growth factor-beta (TGFB) superfamily. Bone AccessionW24850 U.S. morphogenic protein induces bone formation. BMP-2b activitycan be determined Pat. No. 5,631,142 using the following assays known inthe art: Nat Genet. 2001 Jan; 27(1): 84-8; Eur J P12644/BMP4_HUMANBiochem 1996 Apr. 1; 237(1): 295-302; J Biol Cbcre, Vol. 274, Issue 16,10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M.(1996) Genes Dev. 10, 1580-1594. Induction of protein 4 Cartilage,Tissue and Bone Growth, and Diabetes (SEQ ID NO: 171) BMP-4 GeneSeqBMP-4 belongs to the transforming growth factor-beta (TGFB) superfamily.Bone Accession B02796 morphogenic protein induces bone formation. BMP-4activity can be determined WO0020591 using the following assays known inthe art: Nat Genet. 2001 Jan; 27(1): 84-8; Eur J P12644/BMP4_HUMANBiochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16,10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M.(1996) Genes Dev. 10, 1580-1594. protein 4 Induction of Cartilage,Tissue and Bone Growth, and Diabetes (SEQ ID NO: 172) BMP-5 GeneSeqBMP-5 belongs to the transforming growth factor-beta (TGFB) superfamily.Bone Accession B02797 morphogenic protein induces bone formation. BMP-5activity can be determined WO0020591 using the following assays known inthe art: Nat Genet. 2001 Jan; 27(1): 84-8; Eur J P22003/BMP5_HUMANBiochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16,10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M.(1996) Genes Dev. 10, 1580-1594. protein 5 Induction of Cartilage,Tissue and Bone Growth, and Diabetes (isoform 1) (SEQ ID NO: 173)P22003-2|BMP5_HUMAN Isoform 2 of Bone morphogenetic protein 5 (isoform2) (SEQ ID NO: 515) BMP-6 GeneSeq BMP-6 belongs to the transforminggrowth factor-beta (TGFB) superfamily. Bone Accession R32904 U.S.morphogenic protein induces bone formation. BMP-6 activity can bedetermined Pat. No. 5,187,076 using the following assays known in theart: Nat Genet. 2001 Jan; 27(1): 84-8; Eur J P22004/BMP6_HUMAN Biochem1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16,10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M.(1996) Genes Dev. 10, 1580-1594. protein 6 Induction of Cartilage,Tissue and Bone Growth, and Diabetes. Hemochromatosis. (SEQ ID NO: 174)Osteogenic Protein-1; OP- OP-1 belongs to the transforming growthfactor-beta (TGFB) superfamily. Bone 1; BMP-7 GeneSeq morphogenicprotein induces bone formation. OP-1 activity can be determined usingAccession W34783 the following assays known in the art: Nat Genet. 2001Jan; 27(1): 84-8; Eur J WO973462 Biochem 1996 Apr. 1; 237(1): 295-302; JBiol Chem, Vol. 274, Issue 16, 10897-10902, P18075/BMP7_HUMAN Apr. 16,1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Bonemorphogenetic Induction of Cartilage, Tissue and Bone Growth, andDiabetes protein 7 (SEQ ID NO: 175) BMP7 Variant A OP-1 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone (SEQ ID NO:579) morphogenic protein induces bone formation. OP-1 activity can bedetermined using the following assays known in the art: Nat Genet. 2001Jan; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J BiolChem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L.M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue andBone Growth, and Diabetes BMP7 Variant B OP-1 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone (SEQ ID NO:580) morphogenic protein induces bone formation. OP-1 activity can bedetermined using the following assays known in the art: Nat Genet. 2001Jan; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J BiolChem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L.M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue andBone Growth, and Diabetes BMP7 Variant C OP-1 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone (SEQ ID NO:581) morphogenic protein induces bone formation. OP-1 activity can bedetermined using the following assays known in the art: Nat Genet. 2001Jan; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J BiolChem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L.M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue andBone Growth, and Diabetes Osteogenic Protein-2 OP-2 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone GeneSeqAccession morphogenic protein induces bone formation. OP-2 activity canbe determined using R57973 WO9406399 the following assays known in theart: Nat Genet. 2001 Jan; 27(1): 84-8; Eur J P34820/BMP8B_HUMAN Biochem1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16,10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M.(1996) Genes Dev. 0, 1580-1594. protein 8B Induction of Cartilage,Tissue and Bone Growth, and Diabetes (SEQ ID NO: 176) GDF-1 GeneSeqMembers of the TGF-beta family of proteins initiate cell signaling bybinding to Accession R60961 heteromeric receptor complexes of type I(TbetaRI) and type II (TbetaRII) WO9406449 serine/threonine kinasereceptors (reviewed by Massague, J. et al. (1994) TrendsP27539/GDF1_HUMAN Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv.Immunol. 55: 181-220). Embryonic Activation of this heteromeric receptorcomplex occurs when TGF-beta binds to growth/differentiation TbetaRII,which then recruits and phosphorylates TbetaRI. Activated TbetaRI thenfactor 1 propagates the signal to downstream targets (Chen, F. andWeinberg, R. A. (1995) (SEQ ID NO: 177) PNA892: 1565-1569; Wrana, J. L.et al. (1994) Nature 370: 341 347). The effect of GDF-1 on signaling canbe assayed by treating Primary BAECs transferred with a construct calledp3TP-Lux, containing a TGF-beta responsive promoter fused to a reportergene, and measuring luciferase gene expression (Wrana et al., 1994,Nature 370: 341-347). Developmental disorders, Induction of Cartilage,Tissue and Bone Growth, and Diabetes BMP-9 GeneSeq BMP-9 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone AccessionR86903 morphogenic protein induces bone formation. BMP-9 activity can bedetermined WO9533830 using the following assays known in the art: NatGenet. 2001 Jan; 27(1): 84-8; Eur J Q9UK05/GDF2_HUMAN Biochem 1996 Apr.1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902,Growth/differentiation Apr. 16, 1999; and Hogan, B. L. M. (1996) GenesDev. 10, 1580-1594. factor 2 Induction of Cartilage, Tissue and BoneGrowth, and Diabetes (SEQ ID NO: 178) BMP-10 GeneSeq BMP-10 belongs tothe transforming growth factor-beta (TGFB) superfamily. Bone AccessionR66202 morphogenic protein induces bone formation. BMP-10 activity canbe determined WO9426893 using the following assays known in the art: NatGenet. 2001 Jan; 27(1): 84-8; Eur J Q95393/BMP10_HUMAN Biochem 1996 Apr.1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bonemorphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10,1580-1594. protein 10 Induction of Cartilage, Tissue and Bone Growth,and Diabetes (SEQ ID NO: 179) BMP-12 GeneSeq BMP-12 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone AccessionR78734 morphogenic protein induces bone formation. BMP-12 activity canbe determined WO9516035 using the following assays known in the art: NatGenet. 2001 Jan; 27(1): 84-8; Eur J Q7Z4P5/GDF7_HUMAN Biochem 1996 Apr.1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902,Growth/differentiation Apr. 16, 1999; and Hogan, B. L. M. (1996) GenesDev. 10, 1580-1594. factor 7 Induction of Cartilage, Tissue and BoneGrowth, and Diabetes (SEQ ID NO: 180) BMP-15 GeneSeq BMP-15 belongs tothe transforming growth factor-beta (TGFB) superfamily. Bone AccessionW11261 morphogenic protein induces bone formation. BMP-15 activity canbe determined W09636710 using the following assays known in the art: NatGenet. 2001 Jan; 27(1): 84-8; Eur J O95972/BMP15_HUMAN Biochem 1996 Apr.1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bonemorphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10,1580-1594. protein 15 Induction of Cartilage, Tissue and Bone Growth,and Diabetes (SEQ ID NO: 181) BMP-17 GeneSeq BMP-17 belongs to thetransforming growth factor-beta (TGFB) superfamily. Bone AccessionY17870 morphogenic protein induces bone formation. BMP-17 activity canbe determined WO9929718 using the following assays known in the art: NatGenet. 2001 Jan; 27(1): 84-8; Eur J SEQ ID NO: 2 from U.S. Biochem 1996Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902,Pat. No. 7,151,086 Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev.10, 1580-1594. (SEQ ID NO: 182) Induction of Cartilage, Tissue and BoneGrowth, and Diabetes BMP-18 GeneSeq BMP-18 belongs to the transforminggrowth factor-beta (TGFB) superfamily. Bone Accession Y17871 morphogenicprotein induces bone formation. BMP-18 activity can be determinedWO9929718 using the following assays known in the art: Nat Genet. 2001Jan; 27(1): 84-8; Eur J SEQ ID NO: 4 from U.S. Biochem 1996 Apr. 1;237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Pat. No.7,151,086 Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10,1580-1594. (SEQ ID NO: 183) Induction of Cartilage, Tissue and BoneGrowth, and Diabetes Inhibin alpha GeneSeq The inhibin beta A subunitjoins the alpha subunit to form a pituitary FSH secretion AccessionB02806 inhibitor. Inhibin has been shown to regulate gonadal stromalcell proliferation WO0020591 negatively and to have tumor-suppressoractivity. In addition, serum levels of inhibin P05111/INHA_HUMAN havebeen shown to reflect the size of granulosa-cell tumors and cantherefore be Inhibin alpha chain used as a marker for primary as well asrecurrent disease. Tumor suppressor activity (SEQ ID NO: 184) of inhibincan be determined using assays known in the art: Matzuk et al., Nature1992 Nov. 26: 360 (6402); 313-9. Tumor suppression. Inhibin beta GeneSeqThe inhibin beta A subunit joins the alpha subunit to form a pituitaryFSH secretion Accession H02808 inhibitor. Inhibin has been shown toregulate gonadal stromal cell proliferation WO0020591 negatively and tohave tumour-suppressor activity. In addition, serum levels of inhibinP08476/INHBA_HUMAN have been shown to reflect the size of granulosa-celltumors and can therefore be Inhibin beta A chain used as a marker forprimary as well as recurrent disease. Tumor suppressor activity (SEQ IDNO: 185) of inhibin can be determined using assays known in the art:Matzuk et al., Nature P09529/INHBB_HUMAN 1992 Nov. 26: 360 (6402);313-9. Tumor suppression. Inhibin beta B chain (SEQ ID NO: 186) CerberusProtein Cerebus is believed to be involved in the inhibition of BMPactivity BMP activity, in the GeneSeq Accession presence of theantagonist Cerebus, can be determined using the following assays W86032WO9849296 known in the art: Nat Genet. 2001 Jan; 27(1): 84-8; Eur JBiochem 1996 Apr. 1; O95813/CER1_HUMAN 237(1): 295-302; J Biol Chem,Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Cerberus Hogan, B.L. M. (1996) Genes Dev. 10, 1580-1594. BMP Antagonist useful for (SEQ IDNO: 187) Osteosarcoma, abnormal bone growth. Soluble BMP ReceptorSoluble BMP receptor kinase protein-3 is involved in the binding ofBMPs. Soluble Kinase Protein-3 BMP receptor kinase protein-3 is usefulas an antagonist for the inhibition of BMP GeneSeq Accession activity.BMP activity, in the presence of the soluble antagonist BMP receptorkinase R95227 WO9614579 protein-3, can be determined using the followingassays known in the art: Nat Genet. Q13873/BMPR2_HUMAN 2001 Jan; 27(1):84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Bonemorphogenetic Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan,B. L. M. (1996) Genes protein receptor type-2 Dev. 10, 1580-1594. BMPAntagonist useful for Osteosarcoma, abnormal bone (SEQ ID NO: 188)growth. BMP Processing Enzyme BMPs belong to the transforming growthfactor-beta (TGFB) superfamily. Bone Furin GeneSeq Accession morphogenicprotein induces bone formation. BMP activity, in the presence of theW36099 WO9741250 Furin, can be determined using the following assaysknown in the art: Nat Genet. P09958/FURIN_HUMAN 2001 Jan; 27(1): 84-8;Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. Furin 274,Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) GenesDev. (SEQ ID NO: 189) 10, 1580-1594. Bone formation or RegenerationAbnormalities TGF-beta 1 GeneSeq Members of the TGF-beta family ofproteins initiate cell signaling by binding to Accession R29657heteromeric receptor complexes of type I (TbetaRI) and type II(TbetaRII) WO9216228 serine/threonine kinase receptors (reviewed byMassague, J. et al. (1994) Trends P01137/TGFB1_HUMAN Cell Biol. 4: 172178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). Transforminggrowth Activation of this heteromeric receptor complex occurs whenTGF-beta. binds to factor beta-1 TbetaRII, which then recruits andphosphorylates TbetaRI. Activated TbetaRI then (SEQ ID NO: 190)propagates the signal to downstream targets (Chen, F. and Weinberg. R.A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341.The effect of TGF betas on signaling can be assayed by treating PrimaryBAECs transfected with a construct called p3TP-Lux, containing aTGF-beta responsive promoter fused to a reporter gene, and measuringluciferase gene expression (Wrana et al., 1994, Nature 370: 341-347).Useful for treating cancer and to promote wound healing. TGF-beta 2GeneSeq Members of the TGF-beta family of proteins initiate cellsignaling by binding to Accession R39659 heteromeric receptor complexesof type I (TbetaRI) and type II (TbetaRII) EP542679 serine/threoninekinase receptors (reviewed by Massague, J. et al. (1994) TrendsP61812/TGFB2_HUMAN Cell Biol. 4: 172 178; Miyazono, K. et al. (1994)Adv. Immunol. 55: 181-220). Transforming growth Activation of thisheteromeric receptor complex occurs when TGF-beta. binds to factorbeta-2 TbetaRII, which then recruits and phosphorylates TbetaRI.Activated TbetaRI then (SEQ ID NO: 191) propagates the signal todownstream targets (Chen, F. and Weinberg. R. A. (1995) PNA892:1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGFbetas on signaling can be assayed by treating Primary BAECs transfectedwith a construct called p3TP-Lux, containing a TGF-beta responsivepromoter fused to a reporter gene, and measuring luciferase geneexpression (Wrana et al., 1994, Nature 370: 341-347). Useful fortreating cancer and to promote wound healing. ZTGF-beta 9 GeneSeqMembers of the TGF-beta family of proteins initiate cell signaling bybinding to Accession Y70654 heteromeric receptor complexes of type I(TbetaRI) and type II (TbetaRII) WO0015798 serine/threonine kinasereceptors (reviewed by Massague, J. et al. (1994) Trends SEQ ID NO: 2 ofCell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55:181-220). WO0015798 Activation of this heteromeric receptor complexoccurs when TGF-beta. binds to (SEQ ID NO: 192) TbetaRII, which thenrecruits and phosphorylates TbetaRI. Activated TbetaRI then propagatesthe signal to downstream targets (Chen, F. and Weinberg. R. A. (1995)PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. Theeffect of TGF betas on signaling can be assayed by treating PrimaryBAECs transfected with a construct called p3TP-Lux, containing aTGF-beta responsive promoter fused to a reporter gene, and measuringluciferase gene expression (Wrana et al., 1994, Nature 370: 341-347).Useful for treating cancer and to promote wound healing. Anti-TGF betafamily Members of the TGF-beta family of proteins initiate cellsignaling by binding to antibodies GB2305921 heteromeric receptorcomplexes of type I (TbetaRI) and type II (TbetaRII) serine/threoninekinase receptors (reviewed by Massague, J. et al. (1994) Trends CellBiol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220).Activation of this heteromeric receptor complex occurs when TGF-beta.binds to TbetaRII, which then recruits and phosphorylates TbetaRI.Activated TbetaRI then propagates the signal to downstream targets(Chen, F. and Weinberg. R. A. (1995) PNA892: 1565-1569; Wrana, J. L. etal. (1994) Nature 370: 341. The effect of TGF betas on signaling in thepresence of an anti-TGF beta antibody, can be assayed by treatingPrimary BAECs transfected with a construct called p3TP-Lux, containing aTGF-beta responsive promoter fused to a reporter gene, and measuringluciferase gene expression (Wrana et al., 1994, Nature 370: 341-347).Useful for control of fibrosis, immune, and inflammatory disease. LatentTGF beta binding Members of the TGF-beta family of proteins initiatecell signaling by binding to protein II GeneSeq heteromeric receptorcomplexes of type I (TbetaRI) and type II (TbetaRII) Accession Y70552serine/threonine kinase receptors (reviewed by Massague, J. et al.(1994) Trends WO0012551 Cell Biol. 4: 172 178; Miyazono, K. et al.(1994) Adv. Immunol. 55: 181-220). Q14767/LTBP2_HUMAN Activation of thisheteromeric receptor complex occurs when TGF-beta. binds toLatent-transforming TbetaRII, which then recruits and phosphorylatesTbetaRI. Activated TbetaRI then growth factor beta-binding propagatesthe signal to downstream targets (Chen, F. and Weinberg. R. A. (1995)protein 2 PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341.The effect of TGF (SEQ ID NO: 193) betas on signaling in the presence ofa TGF beta binding protein, can be assayed by treating Primary BAECstransfected with a construct called p3TP-Lux, containing a TGF-betaresponsive promoter fused to a reporter gene, and measuring luciferasegene expression (Wrana et al., 1994, Nature 370: 341-347). Useful forinhibiting tissue or tumor growth. MP52 GeneSeq Members of the TGF-betafamily of proteins initiate cell signaling by binding to AccessionW36100 heteromeric receptor complexes of type I (TbetaRI) and type II(TbetaRII) WO9741250 serine/threonine kinase receptors (reviewed byMassague, J. et al. (1994) Trends P43026/GDF5_HUMAN Cell Biol. 4: 172178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220).Growth/differentiation Activation of this heteromeric receptor complexoccurs when TGF-beta. binds to factor 5 TbetaRII, which then recruitsand phosphorylates TbetaRI. Activated TbetaRI then (SEQ ID NO: 194)propagates the signal to downstream targets (Chen, F. and Weinberg. R.A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341.The effect of TGF betas on signaling can be assayed by treating PrimaryBAECs transfected with a construct called p3TP-Lux, containing aTGF-beta responsive promoter fused to a reporter gene, and measuringluciferase gene expression (Wrana et al., 1994, Nature 370: 341-347).Bone formation or Regeneration Abnormalities b57 Protein GeneSeq BMPsare involved in the induction of bone formation. Specific antagonistsare useful Accession W69293 is preventing this activity from occurring.BMP activity, in the presence of b57 protein, WO9837195 can bedetermined using the following assays known in the art: Nat Genet. 2001Jan; SEQ ID NO: 2 of 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1):295-302; J Biol Chem, Vol. 274, Issue WO9837195 16, 1089-10902, Apr. 16,1999; and Hogan, B. L. M. (1996) Genes Deve. 10, 1580-1594. (SEQ ID NO:195) BMP Antagonist useful for Osteosarcoma, abnormal bone growth.Resistin GeneSeq This gene belongs to the family defined by mouse FIZZIand FIZZ3/Resistin genes. Accession W69293 The characteristic feature ofthis family is the C-terminal stretch of 10 cys residues WO0064920 withidentical spacing. The mouse homolog of this protein is secreted byadipocytes, Q9HD89/RETN_HUMAN may be the hormone potentially linkingobesity to type II diabetes. Ability of resistin to Resistin influencetype II diabetes can be determined using assays known in the art:Pontoglio (isoform 1) et al., J Clin Invest 1998 May 15; 101(10):2215-22. Type II diabetes and Syndrome (SEQ ID NO: 196) X. Galectin-4GeneSeq Galectins are a family of carbohydrate-binding proteinscharacterized by an affinity for Accession W11841 beta-galactosidecontaining glycoconjugates. Ability of Galectin-4 polypeptides toWO9703190 bind lactose can be determined using assays known in the art:Wada, et al., J Biol P56470/LEG4_HUMAN Chem 1997 Feb. 28; 272(9):6078-86. Lactose intolerance. Galectin-4 (SEQ ID NO: 197) APM-I;ACRP-30; ACPR30 gene is exclusively expressed in adipose tissue. ACRP30is thought to Famoxin GeneSeq increase fatty acid oxidation by muscletissue. Ability of ACRP30 polypeptides to Accession Y71035 influenceobesity and fat oxidation can be determined using assays known in theart: W00026363 Fruebis et al., Proc Nat'l Acad Sci USA 2001 Feb. 13;98(4): 2005-10. Obesity, Q15848/ADIPO_HUMAN Metabolic disorders, LipidMetabolism; Hormone Secretion. Adiponectin (SEQ ID NO: 198) ACRP-30Homologue; ACPR30 gene is exclusively expressed in adipose tissue.ACRP30 is thought to Complement Component increase fatty acid oxidationby muscle tissue. Ability of ACRP30 homologue Clq C GeneSeq Accessionpolypeptides to influence obesity and fat oxidation can be determinedusing assays B30234 WO0063376 known in the art: Fruebis et al., ProcNat'l Acad Sci USA 2001 Feb. 13; 98(4): 2005-10. P02747/C1QC_HUMANObesity, Metabolic disorders, Lipid Metabolism; Hormone Secretion.Complement C1q subcomponent subunit C (SEQ ID NO: 199) Calpain-10aGeneSeq Calpain is believed to play a role in insulin secretion andinsulin activity, and therefore Accession Y79567 may be useful in thetreatment of type II diabetes. Ability of Calpain-10 to influenceWO0023603 type II diabetes can be determined using assays known in theart: Pontoglio et al., J Q9HC96/CAN10_HUMAN Clin Invest 1998 May 15;101(10): 2215-22. Diabetes mellitus; Regulation of Insulin Calpain-10secretory response; Insulin mediated glucose transport disorders.(Isoform A) (SEQ ID NO: 200) Calpain-10b GeneSeq Calpain is believed toplay a role in insulin secretion and insulin activity, and thereforeAccession Y79568 may be useful in the treatment of type II diabetes.Ability of Calpain-10 to influence WO0023603 type II diabetes can bedetermined using assays known in the art: Pontoglio et al., J Q9HC96-Clin Invest 1998 May 15; 101(10): 2215-22. Diabetes mellitus; Regulationof Insulin 2/CAN10_HUMAN Isoform secretory response; Insulin mediatedglucose transport disorders. B of Calpain-10 (SEQ ID NO: 201)Calpain-10c GeneSeq Calpain is believed to play a role in insulinsecretion and insulin activity, and therefore Accession Y79569 may beuseful in the treatment of type II diabetes. Ability of Calpain-10 toinfluence WO0023603 type II diabetes can be determined using assaysknown in the art: Pontoglio et al., J Q9HC96- Clin Invest 1998 May 15;101(10): 2215-22. Diabetes mellitus; Regulation of Insulin 3/CAN10_HUMANIsoform secretory response; Insulin mediated glucose transportdisorders. C of Calpain-10 (SEQ ID NO: 202) PDGF-D GeneSeq VascularEndothelial Growth Factor. Proliferation assay using NR6R-3T3 cellsAccession Y71130 (Rizzino 1988 Cancer Res. 48: 4266). Wound Healing;Atherosclermis. WO0027879 Q9GZP0/PDGFD_HUMAN Platelet-derived growthfactor D (isoform 1) (SEQ ID NO: 203) FasL GeneSeq Accession Activitiesassociated with apoptosis and immune system functions. Activity can beY28594 WO9936079 determined using Apoptosis assays known in the art:Walczak et al. (1996) EMBOJ P48023/TNFL6_HUMAN 16: 5386-5397.Apoptosis-related disorders; Auto-immune disorders; Graft v-Host Tumornecrosis factor disorders. ligand superfamily member 6 (isoform 1) (SEQID NO: 204) Chondro modulin-like Chondromodulin proteins are cartilageproteins thought to confer resistance to protein GeneSeq anglogeneis,and thus are useful as anti-angiogenic agents that may have utility inAccession Y71262 combating cancer. Ability of Chondromodulin-likeprotein to inhibit vascularization can WO0029579 be determined usingassays known in the art: Hirakie et al., J Biol Chem 1997 Dec. SEQ IDNO: 2 from 19; 272(51): 32419-26. Antianglogenic agent; Osteoblastproliferation stimulator; WO0029579 prevents vascularization ofcartilage tissue; Useful to treat cancer. (SEQ ID NO: 370) PatchedGeneSeq Patched is a tumour-suppressor receptor for Sonic hedgehog(shh), which is a protein Accession W72969 U.S. that controlsdevelopmental patterning and growth. Ability of soluble Patched to bindPat. No. 5,837,538 to and inhibit the activities of shh can bedetermined using assays known in the art: Q13635/PTC1_HUMAN Stone etal., Nature 1996 Nov. 14; 384(6605): 129-34. Receptor for Hedgehogcellular Protein patched homolog 1 proliferation signaling molecule.This receptor is useful as a means of preventing (isoform L) cellularproliferation via the shh signaling path-way, thus useful for cancers.(SEQ ID NO: 205) Patched-2 GeneSeq Patched is a tumour-suppressorreceptor for Sonic hedgehog (shh), which is a protein Accession Y43261that controls developmental patterning and growth. Ability of solublePatched to bind WO9953058 to and inhibit the activities of shh can bedetermined using assays known in the art: Q9Y6C5/PTC2_HUMAN Stone etal., Nature 1996 Nov. 14; 384(6605): 129-34. Receptor for Hedgehogcellular Protein patched homolog 2 proliferation signaling molecule.This receptor is useful as a means of preventing (isoform 1) cellularproliferation via the shh signaling path-way, thus useful for cancers.(SEQ ID NO: 206) Maspin; Protease Inhibitor Maspin is a member of theserpin family of serine protease inhibitors that is thought to 5 GeneSeqAccession suppress tumor metastasis. The inhibitory effects of Maspinand other protease R50938 WO9405804 inhibitors can be assayed usingmethods known in the art such as a labeled protease P36952/SPB5_HUMANsubstrate, for example, Universal Protease Substrate (casein,resorufin-labeled): Serpin B5 Roche Molecular Biochemicals, Cat. No.1080733. Tumor suppressor which is down- (isoform 1) regulated in breastcancers. The maspin protein has tumour suppressing and (SEQ ID NO: 207)invasion suppressing activity. Endostatin GeneSeq Endostatin is believedto inhibit effects of capillary endothelial cell proliferation. TheAccession B28399 inhibitory effects of endostatin can be assayed usingassays disclosed by Cao et al. WO0064946 (1996) J. Biol. Chem. 27129461-29467. Anti-angiogenic activity. Useful in the P39060/COIA1_HUMANprevention and/or treatment of cancers. Collagen alpha-1(XVIII) chain(isoform 1) (SEQ ID NO: 208) aFGF; FGF-1 GeneSeq Fibroblast GrowthFactor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 AccessionP94037 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein.Promotion of growth EP298723 and proliferation of cells, such asepithelial cells and keratinocytes. Antagonists may P05230/FGF1_HUMAN beuseful as anti-cancer agents. Diabetes, Metabolic Disease, Obesity.Fibroblast growth factor 1 (isoform 1) (SEQ ID NO: 209) bFGF; FGF-2GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3cells (Rizzino 1988 Accession R06685 Cancer Res. 48: 4266); Examples 23and 39 disclosed herein. Promotion of growth FR2642086 and proliferationof cells, such as epithelial cells and keratinocytes. Antagonists mayP09038/FGF2_HUMAN be useful as anti-cancer agents. Fibroblast growthfactor 2 (isoform 1) (SEQ ID NO: 210) FGF-3; INT-2 GeneSeq FibroblastGrowth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988Accession R07824 Cancer Res. 48: 4266); Examples 23 and 39 disclosedherein. Promotion of growth WO9503831 and proliferation of cells, suchas epithelial cells and keratinocytes. Antagonists may P11487/FGF3_HUMANbe useful as anti-cancer agents. Fibroblast growth factor 3 (SEQ ID NO:211) FGF-4; HST-1; HBGF-4 Fibroblast Growth Factor Proliferation assayusing NR6R-3T3 cells (Rizzino 1988 GeneSeq Accession Cancer Res. 48:4266); Examples 23 and 39 disclosed herein. Promotion of growth R07825WO9503831 and proliferation of cells, such as epithelial cells andkeratinocytes. Antagonists may P08620/FGF4_HUMAN be useful asanti-cancer agents. Fibroblast growth factor 4 (isoform 1) (SEQ ID NO:212) FGF-5 GeneSeq Fibroblast Growth Factor Proliferation assay usingNR6R-3T3 cells (Rizzino 1988 Accession W22600 Cancer Res. 48: 4266);Examples 23 and 39 disclosed herein. Promotion of growth WO9730155 andproliferation of cells, such as epithelial cells and keratinocytes.Antagonists may P12034/FGF5_HUMAN be useful as anti-cancer agents.Fibroblast growth factor 5 (isoform long) (SEQ ID NO: 213) FGF-6;Heparin binding Fibroblast Growth Factor Proliferation assay usingNR6R-3T3 cells (Rizzino 1988 secreted transforming Cancer Res. 48:4266); Examples 23 and 39 disclosed herein. Promotion of growth factor-2GeneSeq and proliferation of cells, such as epithelial cells andkeratinocytes. Antagonists may Accession R58555 be useful as anti-canceragents. EP613946 P10767/FGF6_HUMAN Fibroblast growth factor 6 (SEQ IDNO: 214) FGF-8 GeneSeq Fibroblast Growth Factor Proliferation assayusing NR6R-3T3 cells (Rizzino 1988 Accession R80783 Cancer Res. 48:4266); Examples 23 and 39 disclosed herein. Promotion of growthWO9524928 and proliferation of cells, such as epithelial cells andkeratinocytes. Antagonists may P55075/FGF8_HUMAN be useful asanti-cancer agents. Fibroblast growth factor 8 (isoform 8E) (SEQ ID NO:215) FGF-9; Gila activating Fibroblast Growth Factor Proliferation assayusing NR6R-3T3 cells (Rizzino 1988 factor GeneSeq Accession Cancer Res.48: 4266); Examples 23 and 39 disclosed herein. Promotion of growthR70822 WO9503831 and proliferation of cells, such as epithelial cellsand keratinocytes. Hair growth. P31371/FGF9_HUMAN Antagonists may beuseful as anti-cancer agents. Fibroblast growth factor 9 (SEQ ID NO:216) FGF-12; Fibroblast growth Fibroblast Growth Factor Proliferationassay using NR6R-3T3 cells (Rizzino 1988 factor homologous factor-1Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion ofgrowth GeneSeq Accession and proliferation of cells, such as epithelialcells and keratinocytes. Antagonists may W06309 WO9635708 be useful asanti-cancer agents. P61328/FGF12_HUMAN Fibroblast growth factor 12(isoform 1) (SEQ ID NO: 217) FGF-19 GeneSeq Fibroblast Growth FactorProliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession Y08582Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion ofgrowth WO9927100 and proliferation of cells, such as epithelial cellsand keratinocytes. Chronic liver O95750/FGF19_HUMAN disease. Primarybiliary cirrhosis. Bile acid-induced liver damage. Antagonists may beFibroblast growth factor 19 useful as anti-cancer agents. (SEQ ID NO:218) FGF-16 GeneSeq Fibroblast Growth Factor Proliferation assay usingNR6R-3T3 cells (Rizzino 1988 Accession Y05474 Cancer Res. 48: 4266);Examples 23 and 39 disclosed herein. Promotion of growth WO9918128 andproliferation of cells, such as epithelial cells and keratinocytes.Antagonists may O43320/FGF16_HUMAN be useful as anti-cancer agents.Fibroblast growth factor 16 (SEQ ID NO: 219) FGF-18 GeneSeq FibroblastGrowth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988Accession Y08590 Cancer Res. 48: 4266); Examples 23 and 39 disclosedherein. Promotion of growth WO9927100 and proliferation of cells, suchas epithelial cells and keratinocytes. Antagonists mayO76093/FGF18_HUMAN be useful as anti-cancer agents. Fibroblast growthfactor 18 (SEQ ID NO: 220) flt-3 ligand GeneSeq Stem Cell ProgenitorChemokine activities can be determined using assays known in AccessionR67541 the art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited EP627487 by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, P49771|FLT3L_HUMAN NJ. Promotionof immune cell growth and/or differentiation. Fms-related tyrosinekinase 3 ligand (isoform 1) (SEQ ID NO: 221) VEGF-110 GeneSeq Promotesthe growth and/or proliferation of endothelial cells. VEGF activity canbe Accession Y69417 determined using assays known in the art, such asthose disclosed in International WO0013702 Publication No. WO0045835,for example. Promotion of growth and proliferation of SEQ ID NO: 11 fromcells, such as vascular endothelial cells. Antagonists may be useful asanti- WO0013702 angiogenic agents, and may be applicable for cancer.(SEQ ID NO: 222) VEGF-121 GeneSeq Promotes the growth and/orproliferation of endothelial cells. VEGF activity can be AccessionB50432 determined using assays known in the art, such as those disclosedin International WO0071713 Publication No. WO0045835, for example.Promotion of growth and proliferation of SEQ ID NO: 2 from cells, suchas vascular endothelial cells. Antagonists may be useful as anti-WO0071713 angiogenic agents, and may be applicable for cancer. (SEQ IDNO: 223) VEGF-138 GeneSeq Promotes the growth and/or proliferation ofendothelial cells. VEGF activity can be Accession Y43483 determinedusing assays known in the art, such as those disclosed in InternationalWO9940197 Publication No. WO0045835, for example. Promotion of growthand proliferation of SEQ ID NO: 4 of cells, such as vascular endothelialcells. Antagonists may be useful as anti- WO99/40197 angiogenic agents,and may be applicable for cancer. (SEQ ID NO: 371) VEGF-145 GeneSeqPromotes the growth and/or proliferation of endothelial cells. VEGFactivity can be Accession Y69413 determined using assays known in theart, such as those disclosed in International WO0013702 Publication No.WO0045835, for example. Promotion of growth and proliferation of SEQ IDNO: 4 from cells, such as vascular endothelial cells. Antagonists may beuseful as anti- WO0013702 angiogenic agents, and may be applicable forcancer. (SEQ ID NO: 224) VEGF-162 GeneSeq Promotes the growth and/orproliferation of endothelial cells. VEGF activity can be AccessionY43484 determined using assays known in the art, such as those disclosedin International W09940197 Publication No. WO0045835, for example.Promotion of growth and proliferation of SEQ ID NO: 8 of cells, such asvascular endothelial cells. Antagonists may be useful as anti-WO99/40197 angiogenic agents, and may be applicable for cancer. (SEQ IDNO: 372) VEGF-165 GeneSeq Promotes the growth and/or proliferation ofendothelial cells. VEGF activity can be Accession Y69414 determinedusing assays known in the art, such as those disclosed in InternationalWO0013702 Publication No. WO0045835, for example. Promotion of growthand proliferation of SEQ ID NO: 6 from cells, such as vascularendothelial cells. Antagonists may be useful as anti- WO0013702angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 225)VEGF-182 GeneSeq Promotes the growth and/or proliferation of endothelialcells. VEGF activity can be Accession Y43483 determined using assaysknown in the art, such as those disclosed in International W09940197Publication No. WO0045835, for example. Promotion of growth andproliferation of SEQ ID NO: 6 of cells, such as vascular endothelialcells. Antagonists may be useful as anti- WO99/40197 angiogenic agents,and may be applicable for cancer. (SEQ ID NO: 373) VEGF-189 GeneSeqPromotes the growth and/or proliferation of endothelial cells. VEGFactivity can be Accession Y69415 determined using assays known in theart, such as those disclosed in International WO0013702 Publication No.WO0045835, for example. Promotion of growth and proliferation of SEQ IDNO: 8 from cells, such as vascular endothelial cells. Antagonists may beuseful as anti- WO0013702 angiogenic agents, and may be applicable forcancer. (SEQ ID NO: 226) VEGF-206 GeneSeq Promotes the growth and/orproliferation of endothelial cells. VEGF activity can be AccessionY69416 determined using assays known in the art, such as those disclosedin International W00013702 Publication No. WO0045835, for example.Promotion of growth and proliferation of SEQ ID NO: 10 from cells, suchas vascular endothelial cells. Antagonists may be useful as anti-WO0013702 angiogenic agents, and may be applicable for cancer. (SEQ IDNO: 227) VEGF-D GeneSeq Promotes the growth and/or proliferation ofendothelial cells. VEGF activity can be Accession W53240 determinedusing assays known in the art, such as those disclosed in InternationalWO9807832 Publication No. WO0045835, for example. Promotion of growthand proliferation of O43915/VEGFD_HUMAN cells, such as vascularendothelial cells. Antagonists may be useful as anti- Vascularendothelial angiogenic agents, and may be applicable for cancer. growthfactor D (SEQ ID NO: 374) VEGF-E; VEGF-X Promotes the growth and/orproliferation of endothelial cells. VEGF activity can be GeneSeqAccession determined using assays known in the art, such as thosedisclosed in International Y33679 WO9947677 Publication No. WO0045835,for example. Promotion of growth and proliferation of SEQ ID NO: 2 fromcells, such as vascular endothelial cells. Antagonists may be useful asanti- WO9947677 angiogenic agents, and may be applicable for cancer.(SEQ ID NO: 228) VEGF Receptor; KDR; flk- Receptor for VEGF polypeptidesVEGF activity, in the presence of flk-1 polypeptides, 1 GeneSeqAccession can be determined using assays known in the art, such as thosedisclosed in W69679 WO9831794 International Publication No. WO0045835,for example. VEGF Receptor. Fusion P35968/VGFR2_HUMAN protein with theextracellular domain is useful as an anti-angiogenic agent. Vascularendothelial Antagonists may be useful in the promotion of angiogenesis.growth factor receptor 2 (isoform 1) (SEQ ID NO: 229) Soluble VEGFReceptor Receptor for VEGF polypeptides VEGF activity, in the presenceof VEGF Receptor GeneSeq Accession polypeptides, can be determined usingassays known in the art, such as those W47037 U.S. Pat. No. disclosed inInternational Publication No. WO0045835, for example. VEGF Receptor.5,712,380 Fusion protein with the extracellular domain is useful as ananti-angiogenic agent. sVEGF-RI (FIG. 3) of Antagonists may be useful inthe promotion of angiogenesis. U.S. Pat. No. 5,712,380 (SEQ ID NO: 442)sVEGF-RII (FIG. 11) of U.S. Pat. No. 5,712,380 (SEQ ID NO: 443)sVEGF-RTMI (FIG. 15) of U.S. Pat. No. 5,712,380 (SEQ ID NO: 444)sVEGF-RTMII (FIG. 13) of U.S. Pat. No. 5,712,380 (SEQ ID NO: 445) flt-1GeneSeq Accession Receptor for VEGF polypeptides VEGF activity, in thepresence of flt-1 polypeptides, Y70751 WO0021560 can be determined usingassays known in the art, such as those disclosed in P17948/VGFR1_HUMANInternational Publication No. WO0045835, for example. VEGF Receptor.Fusion Vascular endothelial protein with the extracellular domain isuseful as an anti-angiogenic agent. growth factor receptor 1 Antagonistsmay be useful in the promotion of angiogenesis. (isoform 1) (SEQ ID NO:230) VEGF R-3; flt-4 GeneSeq Receptor for VEGF polypeptides VEGFactivity, in the presence of flt-4 polypeptides, Accession B29047 can bedetermined using assays known in the art, such as those disclosed inWO0058511 International Publication No. WO0045835, for example. VEGFReceptor. Fusion P35916/VGFR3_HUMAN protein with the extracellulardomain is useful as an anti-angiogenic agent. Vascular endothelialAntagonists may be useful in the promotion of angiogenesis. growthfactor receptor 3 (isoform 1) (SEQ ID NO: 231) Neuropilin-1 GeneSeqVascular Endothelial Growth Factor VEGF activity can be determined usingassays Accession Y06319 known in the art, such as those disclosed inInternational Publication No. WO9929858 WO0045835, for example.Promotion of growth and proliferation of cells, such asO14786/NRP1_HUMAN vascular endothelial cells. Antagonists may be usefulas anti-angiogenic agents, and Neuropilin-1 may be applicable forcancer. (isoform 1) (SEQ ID NO: 232) Neuropilin-2 GeneSeq VascularEndothelial Growth Factor VEGF activity can be determined using assaysAccession Y03618 known in the art, such as those disclosed inInternational Publication No. WO9929858 WO0045835, for example.Promotion of growth and proliferation of cells, such asO60462/NRP2_HUMAN vascular endothelial cells. Antagonists may be usefulas anti-angiogenic agents, and Neuropilin-2 may be applicable forcancer. (isoform A22) (SEQ ID NO: 233) Human fast twitch skeletalTroponins are contractile proteins that are thought to inhibitangiogenesis. High levels muscle troponin C may contribute to thedifficulty encountered in revascularizing the ischemic GeneSeq Accessionmyocardium after cardiovascular injury. Ability of soluble Troponins toinhibit W22597 W09730085 angiogenesis can be determined using assaysknown in the art:. Proc Natl Acad Sci P02585/TNNC2_HUMAN USA 1999 Mar.16; 96(6): 2645-50. Anti-angiogenesis Troponin C, skeletal muscle (SEQID NO: 234) Human fast twitch skeletal Troponins are contractileproteins that are thought to inhibit angiogenesis. High levels muscletroponin I may contribute to the difficulty encountered inrevascularizing the ischemic GeneSeq Accession myocardium aftercardiovascular injury. Ability of soluble Troponins to inhibit W18054W09730085 angiogenesis can be determined using assays known in the art.Proc Natl Acad Sci P48788/TNNI2_HUMAN USA 1999 Mar. 16; 96(6): 2645-50.Anti-angiogenesis Troponin I, fast skeletal muscle (isoform 1) (SEQ IDNO: 235) Human fast twitch skeletal Troponins are contractile proteinsthat are thought to inhibit angiogenesis. High levels muscle troponin Tmay contribute to the difficulty encountered in revascularizing theischemic GeneSeq Accession myocardium after cardiovascular injury.Ability of soluble Troponins to inhibit W22599 W09730085 angiogenesiscan be determined using assays known in the art:. Proc Natl Acad Sci SEQID NO: 3 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesisWO9730085 (SEQ ID NO: 236) fragment. myofibrillar Troponins arecontractile proteins that are thought to inhibit angiogenesis. Highlevels protein troponin I may contribute to the difficulty encounteredin revascularizing the ischemic GeneSeq Accession myocardium aftercardiovascular injury. Ability of soluble Troponins to inhibit W18053W09719955 angiogenesis can be determined using assays known in the art:.Proc Natl Acad Sci SEQ ID NO: 3 of USA 1999 Mar. 16; 96(6): 2645-50.Anti-angiogenesis WO9719955 (SEQ ID NO: 237) myofibrillar proteinTroponins are contractile proteins that are thought to inhibitangiogenesis. High levels troponin I GeneSeq may contribute to thedifficulty encountered in revascularizing the ischemic Accession W18054myocardium after cardiovascular injury. Ability of soluble Troponins toinhibit WO9719955 angiogenesis can be determined using assays known inthe art:. Proc Natl Acad Sci SEQ ID NO: 3 of USA 1999 Mar. 16; 96(6):2645-50. Anti-angiogenesis WO9719955 (SEQ ID NO: 237) Troponin peptidesTroponins are contractile proteins that are thought to inhibitangiogenesis. High levels GeneSeq Accessions may contribute to thedifficulty encountered in revascularizing the ischemic Y29581, Y29582,Y29583, myocardium after cardiovascular injury. Ability of solubleTroponins to inhibit Y29584, Y29585, and angiogenesis can be determinedusing assays known in the art:. Proc Natl Acad Sci Y29586 WO9933874 USA1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis Wildtype troponinsprovided as: Human fast twitch skeletal muscle troponin C GeneSeqAccession W22597 WO9730085 P02585/TNNC2_HUMAN Troponin C, skeletalmuscle (SEQ ID NO: 234) Human fast twitch skeletal muscle troponin IGeneSeq Accession W18054 W09730085 P48788/TNNI2_HUMAN Troponin I, fastskeletal muscle (isoform 1) (SEQ ID NO: 235) Human fast twitch skeletalmuscle troponin T GeneSeq Accession W22599 W09730085 SEQ ID NO: 3 ofWO9730085 (SEQ ID NO: 236) fragment. myofibrillar protein troponin IGeneSeq Accession W18053 W09719955 SEQ ID NO: 3 of WO9719955 (SEQ ID NO:237) Human fast twitch skeletal muscle Troponin subunit C GeneSeqAccession B00134 WO0054770 SEQ ID NO: 1 of WO0054770 (SEQ ID NO: 375)Human fast twitch skeletal muscle Troponin subunit I Protein GeneSeqAccession B00135 WO0054770 SEQ ID NO: 2 of WO0054770 (SEQ ID NO: 376)Human fast twitch skeletal muscle Troponin subunit T GeneSeq AccessionB00136 WO0054770 SEQ ID NO: 3 of WO0054770 (SEQ ID NO: 377) Human fasttwitch skeletal Troponins are contractile proteins that are thought toinhibit angiogenesis. High levels muscle Troponin subunit C maycontribute to the difficulty encountered in revascularizing the ischemicGeneSeq Accession myocardium after cardiovascular injury. Ability ofsoluble Troponins to inhibit B00134 WO0054770 angiogenesis can bedetermined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO:1 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO0054770 (SEQID NO: 375) Human fast twitch skeletal Troponins are contractileproteins that are thought to inhibit angiogenesis. High levels muscleTroponin subunit I may contribute to the difficulty encountered inrevascularizing the ischemic Protein GeneSeq myocardium aftercardiovascular injury. Ability of soluble Troponins to inhibit AccessionB00135 angiogenesis can be determined using assays known in the art:.Proc Natl Acad Sci WO0054770 USA 1999 Mar. 16; 96(6): 2645-50.Anti-angiogenesis SEQ ID NO: 2 of WO0054770 (SEQ ID NO: 376) Human fasttwitch skeletal Troponins are contractile proteins that are thought toinhibit angiogenesis. High levels muscle Troponin subunit T maycontribute to the difficulty encountered in revascularizing the ischemicGeneSeq Accession myocardium after cardiovascular injury. Ability ofsoluble Troponins to inhibit B00136 WO0054770 angiogenesis can bedetermined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO:3 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO0054770 (SEQID NO: 377) Activator Inbibitor-1; PAI-1 PAIs are believed to play arole in cancer, and cardiovascular disease and blood- GeneSeq Accessionclotting disorders. Methods that measure plasminogen activator inhibitor(PAI) activity R08411 WO9013648 are known in the art, for example, assaythe ability of PAI to inhibit tissue P05121/PAI1_HUMAN plasminogenactivator (tPA) or urokinase (uPA): J Biochem Biophys Methods 2000Plasminogen activator Sep. 11; 45(2): 127-40, Breast Cancer Res Treat1996; 41(2): 141-6. Methods that inhibitor 1 measure anti-angiogenesisactivity are known in the art, for example, Proc Natl Acad (isoform 1)Sci USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis; blood-clottingdisorders. (SEQ ID NO: 238) Plasminogen Activator PAIs are believed toplay a role in cancer, and cardiovascular disease and blood-Inhibitor-2; PAI-2 clotting disorders. Methods that measure plasminogenactivator inhibitor (PAI) activity GeneSeq Accession are known in theart, for example, assay the ability of PAI to inhibit tissue P94160DE3722673 plasminogen activator (tPA) or urokinase (uPA): J BiochemBiophys Methods 2000 P05120/PAI2_HUMAN Sep. 11; 45(2): 127-40, BreastCancer Res Treat 1996; 41(2): 141-6. Methods that Plasminogen activatormeasure anti-angiogenesis activity are known in the art, for example,Proc Natl Acad inhibitor 2 Sci USA 1999 Mar. 16; 96(6): 2645-50.Anti-angiogenesis; blood-clotting disorders. (SEQ ID NO: 239) ActivatorInhibitor-2; PAI-2 PAIs are believed to play a role in cancer, andcardiovascular disease and blood- GeneSeq Accession clotting disorders.Methods that measure plasminogen activator inhibitor (PAI) activityR10921 WO9102057 are known in the art, for example, assay the ability ofPAI to inhibit tissue P05120/PAI2_HUMAN plasminogen activator (tPA) orurokinase (uPA): J Biochem Biophys Methods 2000 Plasminogen activatorSep. 11; 45(2): 127-40, Breast Cancer Res Treat 1996; 41(2): 141-6.Methods that inhibitor 2 measure anti-angiogenesis activity are known inthe art, for example, Proc Natl Acad (SEQ ID NO: 239) Sci USA 1999 Mar.16; 96(6): 2645-50. Anti-angiogenesis; blood-clotting disorders. HumanPAI-1 mutants PAIs are believed to play a role in cancer, andcardiovascular disease and blood- GeneSeq Accessions clotting disorders.Methods that measure plasminogen activator inhibitor (PAI) activityR11755, R11756, R11757, are known in the art, for example, assay theability of PAI to inhibit tissue R11758, R11759, R11760, plasminogenactivator (tPA) or urokinase (uPA): J Biochem Biophys Methods 2000R11761, R11762 and Sep. 11; 45(2): 127-40, Breast Cancer Res Treat 1996;41(2): 141-6. Methods that R11763 WO9105048 measure anti-angiogenesisactivity are known in the art, for example, Proc Natl Acad WildtypePAI-1 is provided Sci USA 1999 Mar. 16; 96(6): 2645-50.Anti-angiogenesis; blood-clotting disorders. as P05121/PAI1_HUMANPlasminogen activator inhibitor 1 (isoform 1) (SEQ ID NO: 238) CXCR3;CXC GeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession Y79372 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO0018431Members of this family are involved in a similarly diverse range ofpathologies P49682|CXCR3_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C—X—C chemokinereceptor chemokines exert their effects by acting on a family of seventransmembrane G- type 3 protein coupled receptors. Over 40 humanchemokines have been described, which (isoform 1) bind to ~17 receptorsthus far identified. Chemokine activities can be determined (SEQ ID NO:240) using assays known in the art: Methods in Molecular Biology, 2000,vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble CXCR3polypeptides may be useful for inhibiting chemokine activities and viralinfection. Modified Rantes GeneSeq Chemokines are a family of relatedsmall, secreted proteins involved in biological Accession W38129processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. WO9737005 Members of this family are involved in asimilarly diverse range of pathologies Wildtype Rantes providedincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The herein as chemokines exert their effects by acting ona family of seven transmembrane G- P13501/CCL5_HUMAN C- protein coupledreceptors. Over 40 human chemokines have been described, which C motifchemokine 5 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 241) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders. RANTES GeneSeq Chemokines area family of related small, secreted proteins involved in biologicalAccession Y05299 processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. EP905240 Members of this family are involved in asimilarly diverse range of pathologies P13501/CCL5_HUMAN C- includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The C motif chemokine 5 chemokines exert their effects byacting on a family of seven transmembrane G- (SEQ ID NO: 241) proteincoupled receptors. Over 40 human chemokines have been described, whichbind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders. MCP-Ia GeneSeq Chemokines are a family of related small,secreted proteins involved in biological Accession R73914 processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.WO9509232 Members of this family are involved in a similarly diverserange of pathologies MCP-1 provided as including inflammation, allergy,tissue rejection, viral infection, and tumor biology. TheP13500/CCL2_HUMAN C- chemokines exert their effects by acting on afamily of seven transmembrane G- C motif chemokine 2 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 337) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune disorders. MCP-Ib GeneSeq Chemokines are a family of relatedsmall, secreted proteins involved in biological Accession Y26176processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. WO9929728 Members of this family are involved in asimilarly diverse range of pathologies MCP-1 provided as includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The P13500/CCL2_HUMAN C- chemokines exert their effects byacting on a family of seven transmembrane G- C motif chemokine 2 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 337) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Immune disorders. MCP-I receptor GeneSeq Chemokines are afamily of related small, secreted proteins involved in biologicalAccession R79165 processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. WO9519436 Members of this family are involved ina similarly diverse range of pathologies MCP-IA including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The SEQID NO: 2 of chemokines exert their effects by acting on a family ofseven transmembrane G- WO9519436 protein coupled receptors. Over 40human chemokines have been described, which (SEQ ID NO: 446) bind to ~17receptors thus far identified. Chemokine activities can be determinedMCP-1B using assays known in the art: Methods in Molecular Biology,2000, vol. 138: SEQ ID NO: 4 of Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. WO9519436 Humana Press Inc.,Totowa, NJ. Soluble MCP-1 Receptor polypeptides may be (SEQ ID NO: 447)useful for inhibiting chemokine activities and viral infection. MCP-3GeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession R73915 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W09509232Members of this family are involved in a similarly diverse range ofpathologies P80098/CCL7_HUMAN C- including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C motif chemokine 7chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 336) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MCP-4receptor GeneSeq Chemokines are a family of related small, secretedproteins involved in biological Accession W56689 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO9809171Members of this family are involved in a similarly diverse range ofpathologies SEQ ID NO: 2 of including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The WO9809171 chemokinesexert their effects by acting on a family of seven transmembrane G- (SEQID NO: 378) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Soluble MCP-4 Receptor polypeptides may beuseful for inhibiting chemokine activities and viral infection. RANTESreceptor Chemokines are a family of related small, secreted proteinsinvolved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W29588 U.S. Pat.No. Members of this family are involved in a similarly diverse range ofpathologies 5,652,133 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 2 of U.S. Pat.chemokines exert their effects by acting on a family of seventransmembrane G- No. 5,652,133 protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 379) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble RANTES Receptorpolypeptides may be useful for inhibiting chemokine activities and viralinfection. CCR5 variant GeneSeq Chemokines are a family of relatedsmall, secreted proteins involved in biological Accession W88238processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. WO9854317 Members of this family are involved in asimilarly diverse range of pathologies Variants of wildtype CCR5including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The which has the sequence chemokines exert their effectsby acting on a family of seven transmembrane G- of: protein coupledreceptors. Over 40 human chemokines have been described, whichP51681|CCR5_HUMAN bind to ~17 receptors thus far identified. Chemokineactivities can be determined C-C chemokine receptor using assays knownin the art: Methods in Molecular Biology, 2000, vol. 138: type 5Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. (SEQ ID NO: 448) Humana Press Inc., Totowa, NJ. SolubleCCR5 polypeptides may be useful for inhibiting chemokine activities andviral infection. CCR7 GeneSeq Chemokines are a family of related small,secreted proteins involved in biological Accession B50859 U.S. processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.Pat. No. 6,153,441 Members of this family are involved in a similarlydiverse range of pathologies P32248/CCR7_HUMAN including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The C-Cchemokine receptor chemokines exert their effects by acting on a familyof seven transmembrane G- type 7 protein coupled receptors. Over 40human chemokines have been described, which (SEQ ID NO: 243) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble CCR7polypeptides may be useful for inhibiting chemokine activities and viralinfection. CXC3 GeneSeq Chemokines are a family of related small,secreted proteins involved in biological Accession W23345 processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.WO9727299 Members of this family are involved in a similarly diverserange of pathologies P78423/X3CL1_HUMAN including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The Fractalkinechemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 244) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. EotaxinGeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession W10099 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO9700960Members of this family are involved in a similarly diverse range ofpathologies P51671/CCL11_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The Eotaxin chemokinesexert their effects by acting on a family of seven transmembrane G- (SEQID NO: 245) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders. Neurotactin GeneSeqNeurotactin may play a role in chemotactic leukocyte migration and brainAccessions Y77537, inflammation processes. Chemotactic leukocytemigration assays are known in the W34307, Y53259, and, art, for example:J. Immunol. Methods 33, ((1980)); Nature 1997 Jun. 5; 387(6633): Y77539U.S. Pat. No. 611-7. Immune disorders. 6,013,257 WO9742224P78423/X3CL1_HUMAN Fractalkine (SEQ ID NO: 244) Human CKbeta-9Chemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. B50860 U.S. Pat. No. Members ofthis family are involved in a similarly diverse range of pathologies6,153,441 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The SEQ ID NO: 2 of U.S. Pat. chemokinesexert their effects by acting on a family of seven transmembrane G- No.6,153,441 protein coupled receptors. Over 40 human chemokines have beendescribed, which (SEQ ID NO: 246) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Immune disorders. LymphotactinGeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession B50052 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO0073320Members of this family are involved in a similarly diverse range ofpathologies P47992/XCL1_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The Lymphotactinchemokines exert their effects by acting on a family of seventransmembrane G. (SEQ ID NO: 247) Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MIP-3alpha GeneSeq Chemokines are a family of related small, secretedproteins involved in biological Accession W44398 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO9801557Members of this family are involved in a similarly diverse range ofpathologies P78556/CCL20_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C-C motif chemokine20 chemokines exert their effects by acting on a family of seventransmembrane G. (isoform 1) Chemokine activities can be determinedusing assays known in the art: Methods in (SEQ ID NO: 248) MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune disorders. MIP-3 beta GeneSeq Chemokines are a family ofrelated small, secreted proteins involved in biological Accession W44399processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. WO9801557 Members of this family are involved in asimilarly diverse range of pathologies Q99731/CCL19_HUMAN includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The C-C motif chemokine 19 chemokines exert their effects byacting on a family of seven transmembrane G. (SEQ ID NO: 249) Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Immune disorders. MIP-Gamma GeneSeq Chemokines are a familyof related small, secreted proteins involved in biological AccessionR70798 processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. WO2006135382 Members of this family are involved in asimilarly diverse range of pathologies (SEQ ID NO: 457) includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The chemokines exert their effects by acting on a family ofseven transmembrane G. Chemokine activities can be determined usingassays known in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. Stem CellInhibitory Factor Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. R11553 WO9104274Members of this family are involved in a similarly diverse range ofpathologies SCIF in Table I of including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The WO9104274 chemokinesexert their effects by acting on a family of seven transmembrane G. (SEQID NO: 380) Chemokine activities can be determined using assays known inthe art: Methods in SCIF in Table II of Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, WO9104274 T. N.C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. (SEQ ID NO:381) Hematopoietic growth factors. Thrombopoietin GeneSeq Thrombopoietinis involved in the regulation of the growth and differentiation ofAccession R79905 megakaryocytes and preceptors thereof. Thrombopoietin(TPO) can be assayed to WO9521920 determine regulation of growth anddifferentiation of megakaryocytes. Mol Cell Biol P40225|TPO_HUMAN 2001Apr; 21(8): 2659-70; Exp Hematol 2001 Jan; 29(1): 51-8 and within.Thrombopoietin Hematopoietic growth factors. (isoform 1) (SEQ ID NO:250) c-kit ligand; SCF; Mast cell C-kit ligan is thought to stimulatethe proliferation of mast cells, and is able to growth factor; MGF;augment the proliferation of both myeloid and lymphoid hematopoieticprogenitors in Fibrosarcoma-derived bone marrow culture. C-kit ligand isalso though to act synergistically with other stem cell factor GeneSeqcytokines. Chemokine activities can be determined using assays known inthe art: Accession Y53284, Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, R83978 and R83977 T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. EP992579and EP676470 Hematopoietic growth factors. P21583|SCF_HUMAN Kit ligand(isoform 1) (SEQ ID NO: 251) Platelet derived growth VascularEndothelial Growth Factor VEGF activity can be determined using assaysfactor GeneSeq Accession known in the art, such as those disclosed inInternational Publication No. B48653 WO0066736 WO0045835, for example.Promotion of growth and proliferation of cells, such as PDGF-A vascularendothelial cells. Antagonists may be useful as anti-angiogenic agents,and P04085/PDGFA_HUMAN may be applicable for cancer. Platelet-derivedgrowth factor subunit A (Isoform long) (SEQ ID NO: 257) PDGF-BP01127/PDGFB_HUMAN Platelet-derived growth factor subunit B (isoform 1)(SEQ ID NO: 258) Melanoma inhibiting Melanoma inhibiting protein hasmelanoma-inhibiting activity and can be used to treat protein GeneSeqcancer (melanoma, glioblastoma, neuroblastoma, small cell lung cancer,Accession R69811 neuroectodermal tumors) or as an immunosuppressant (itinhibits IL-2 or WO9503328 phytohaemagglutinin induced proliferation ofperipheral blood lymphocytes. Tumor (SEQ ID NO: 458) suppressor activityof melanoma inhibiting protein can be determined using assays known inthe art: Matzuk et al., Nature 1992 Nov. 26; 360(6402): 313-9. Cancer;melanoma Glioma-derived growth Vascular Endothelial Growth Factor VEGFactivity can be determined using assays factor GeneSeq Accession knownin the art, such as those disclosed in International Publication No.R08120 EP399816 WO0045835, for example. Promotion of growth andproliferation of cells, such as vascular endothelial cells. Antagonistsmay be useful as anti-angiogenic agents, and may be applicable forcancer. Platelet derived growth Vascular Endothelial Growth Factor VEGFactivity can be determined using assays factor precursor A known in theart, such as those disclosed in International Publication No. GeneSeqAccession WO0045835, for example. Promotion of growth and proliferationof cells, such as R84759 EP682110 vascular endothelial cells.Antagonists may be useful as anti-angiogenic agents, and PDGF-Aprecursor (variant may be applicable for cancer. D1) (SEQ ID NO: 382)PDGF-A precursor (variant 13-1) (SEQ ID NO: 383) Platelet derived growthVascular Endothelial Growth Factor VEGF activity can be determined usingassays factor precursor B known in the art, such as those disclosed inInternational Publication No. GeneSeq Accession WO0045835, for example.Promotion of growth and proliferation of cells, such as R84760 EP682110,FIG. vascular endothelial cells. Antagonists may be useful asanti-angiogenic agents, and 1 or FIG. 2 may be applicable for cancer.Wildtype PDGF-B provided as: PDGF-B P01127/PDGFB_HUMAN Platelet-derivedgrowth factor subunit B (isoform 1) (SEQ ID NO: 258) Platelet derivedgrowth Vascular Endothelial Growth Factor VEGF activity can bedetermined using assays factor Bvsis GeneSeq known in the art, such asthose disclosed in International Publication No. Accession P80595 andWO0045835, for example. Promotion of growth and proliferation of cells,such as P80596 EP282317 vascular endothelial cells. Antagonists may beuseful as anti-angiogenic agents, and FIG. 1 of EP282317 may beapplicable for cancer. (SEQ ID NO: 384) Placental Growth Factor VascularEndothelial Growth Factor VEGF activity can be determined using assaysGeneSeq Accessions known in the art, such as those disclosed inInternational Publication No. R23059 and R23060 WO0045835, for example.Promotion of growth and proliferation of cells, such as WO9206194vascular endothelial cells. Antagonists may be useful as anti-angiogenicagents, and P49763-2/PLGF_HUMAN may be applicable for cancer. IsoformPIGF-1 of Placenta growth factor (isoform PIGF-1) (SEQ ID NO: 252)Placental Growth Factor-2 Vascular Endothelial Growth Factor VEGFactivity can be determined using assays GeneSeq Accession known in theart, such as those disclosed in International Publication No. Y08289DE19748734 WO0045835, for example. Promotion of growth and proliferationof cells, such as P49763-3/PLGF_HUMAN vascular endothelial cells.Antagonists may be useful as anti-angiogenic agents, and Isoform PIGF-2of may be applicable for cancer. Placenta growth factor (isoform PIGF-2)(SEQ ID NO: 253) Thrombopoietin Thrombopoietin is involved in theregulation of the growth and differentiation of derivative1 GeneSeqmegakaryocytes and preceptors thereof. Thrombopoietin (TPO) can beassayed to Accession Y77244 determine regulation of growth anddifferentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table3) 2001 Apr; 21(8): 2659-70; Exp Hematol 2001 Jan; 29(1): 51-8 andwithin. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as:P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250)Thrombopoietin Thrombopoietin is involved in the regulation of thegrowth and differentiation of derivative2 GeneSeq megakaryocytes andpreceptors thereof. Thrombopoietin (TPO) can be assayed to AccessionY77255 determine regulation of growth and differentiation ofmegakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 Apr; 21(8):2659-70; Exp Hematol 2001 Jan; 29(1): 51-8 and within. Wildtypethrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMANThrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivativeThrombopoietin is involved in the regulation of the growth anddifferentiation of 3 GeneSeq Accession megakaryocytes and preceptorsthereof. Thrombopoietin (TPO) can be assayed to Y77262 determineregulation of growth and differentiation of megakaryocytes. Mol CellBiol WO0000612 (e.g. Table 3) 2001 Apr; 21(8): 2659-70; Exp Hematol 2001Jan; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia,cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ IDNO: 250) Thrombopoietin derivative Thrombopoietin is involved in theregulation of the growth and differentiation of 4 GeneSeq Accessionmegakaryocytes and preceptors thereof. Thrombopoietin (TPO) can beassayed to Y77267 determine regulation of growth and differentiation ofmegakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 Apr; 21(8):2659-70; Exp Hematol 2001 Jan; 29(1): 51-8 and within. Wildtypethrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMANThrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivativeThrombopoietin is involved in the regulation of the growth anddifferentiation of 5 GeneSeq Accession megakaryocytes and preceptorsthereof. Thrombopoietin (TPO) can be assayed to Y77246 determineregulation of growth and differentiation of megakaryocytes. Mol CellBiol WO0000612 (e.g. Table 3) 2001 Apr; 21(8): 2659-70; Exp Hematol 2001Jan; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia,cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ IDNO: 250) Thrombopoietin derivative Thrombopoietin is involved in theregulation of the growth and differentiation of 6 GeneSeq Accessionmegakaryocytes and preceptors thereof. Thrombopoietin (TPO) can beassayed to Y77253 determine regulation of growth and differentiation ofmegakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 Apr; 21(8):2659-70; Exp Hematol 2001 Jan; 29(1): 51-8 and within. Wildtypethrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMANThrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivativeThrombopoietin is involved in the regulation of the growth anddifferentiation of 7 GeneSeq Accession megakaryocytes and preceptorsthereof. Thrombopoietin (TPO) can be assayed to Y77256 determineregulation of growth and differentiation of megakaryocytes. Mol CellBiol WO0000612 (e.g. Table 3) 2001 Apr; 21(8): 2659-70; Exp Hematol 2001Jan; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia,cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ IDNO: 250) Fractalkine GeneSeq Fractalkine is believed to play a role inchemotactic leukocyte migration and Accession Y53255 U.S. neurologicaldisorders. Fractalkine activity can be determined using Chemotactic Pat.No. 6,043,086 leukocyte migration assays known in the art, for example:J. Immunol. Methods 33, P78423/X3CL1_HUMAN ((1980)); Nature 1997 Jun. 5;387(6633): 611-7. Immune disorders. Fractalkine (SEQ ID NO: 244) CXC3GeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession W23345 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO9757599Members of this family are involved in a similarly diverse range ofpathologies P78423/X3CL1_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The Fractalkinechemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 244) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. CCR7GeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession B50859 U.S. processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Pat. No.6,153,441 Members of this family are involved in a similarly diverserange of pathologies P32248/CCR7_HUMAN including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The C-C chemokinereceptor chemokines exert their effects by acting on a family of seventransmembrane G- type 7 protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 243) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble CCR7polypeptides may be useful for inhibiting chemokine activities and viralinfection. Nerve Growth Factor-beta Nerve Growth Factor Proliferationassay using NR6R-3T3 cells (Rizzino 1988 Cancer GeneSeq Accession Res.48: 4266) Neurological disorders, cancer R11474 EP414151P01138/NGF_HUMAN Beta-nerve growth factor (SEQ ID NO: 254) Nerve GrowthFactor- Nerve Growth Factor Proliferation assay using NR6R 3T3 cells(Rizzino 1988 Cancer beta2 GeneSeq Accession Res. 48: 4266 Neurologicaldisorders, cancer W69725 EP859056 FIG. 1 of EP859056 (SEQ ID NO: 465)Neurotrophin-3 GeneSeq Neurotrophins regulate neuronal cell survival andsynaptic plasticity. Trk tyrosine Accession W8889 kinase activationassays known in the art can be used to assay for neurotrophin WO9821234activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6):3555-3560. P20783/NTF3_HUMAN Neurological disorders, cancerNeurotrophin-3 (isoform 1) (SEQ ID NO: 255) Neurotrophin-4 GeneSeqNeurotrophins regulate neuronal cell survival and synaptic plasticity.Trk tyrosine Accession R47100 kinase activation assays known in the artcan be used to assay for neurotrophin WO9325684 activity, for example,Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. P34130/NTF4_HUMANNeurological disorders, cancer Neurotrophin-4 (SEQ ID NO: 256)Neurotrophin-4a Neurotrophins regulate neuronal cell survival andsynaptic plasticity. Trk tyrosine GeneSeq Accession kinase activationassays known in the art can be used to assay for neurotrophin R47101WO9325684 activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13;98(6): 3555-3560. Wildtype neurotrophin Neurological disorders, cancerprovided as: P34130/NTF4_HUMAN Neurotrophin-4 (SEQ ID NO: 256)Neurotrophin-4b Neurotrophins regulate neuronal cell survival andsynaptic plasticity. tyrosine kinases. GeneSeq Accession Trk tyrosinekinase activation assays known in the art can be used to assay forR47102 WO9325684 neurotrophin activity, for example, Proc Natl Acad SciUSA 2001 Mar. 13; 98(6): 3555-3560. P34130/NTF4_HUMAN Neurologicaldisorders, cancer Neurotrophin-4 (SEQ ID NO: 256) Neurotrophin-4cNeurotrophins regulate neuronal cell survival and synaptic plasticity.tyrosine kinases. GeneSeq Accession Trk tyrosine kinase activationassays known in the art can be used to assay for R47103 WO9325684neurotrophin activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13;98(6): 3555-3560. P34130/NTF4_HUMAN Neurological disorders, cancerNeurotrophin-4 (SEQ ID NO: 256) Neurotrophin-4d Neurotrophins regulateneuronal cell survival and synaptic plasticity. tyrosine kinases.GeneSeq Accession Trk tyrosine kinase activation assays known in the artcan be used to assay for R47102 WO9325684 neurotrophin activity, forexample, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560.P34130/NTF4_HUMAN Neurological disorders, cancer Neurotrophin-4 (SEQ IDNO: 256) Platelet-Derived Growth Vascular Endothelial Growth Factor VEGFactivity can be determined using assays Factor A chain GeneSeq known inthe art, such as those disclosed in International Publication No.Accession R38918 U.S. W00045835, for example. Promotion of growth andproliferation of cells, such as Pat. No. 5,219,739 vascular endothelialcells. Hematopoietic and immune dis-orders. Antagonists mayP04085/PDGFA_HUMAN be useful as anti-angiogenic agents, and may beapplicable for cancer Platelet-derived growth factor subunit A (Isoformlong) (SEQ ID NO: 257) Platelet-Derived Growth Vascular EndothelialGrowth Factor VEGF activity can be determined using assays Factor Bchain GeneSeq known in the art, such as those disclosed in InternationalPublication No. Accession R38919 U.S. W00045835, for example. Promotionof growth and proliferation of cells, such as Pat. No. 5,219,739vascular endothelial cells. Hematopoietic and immune dis-orders.Antagonists may P01127/PDGFB_HUMAN be useful as anti-angiogenic agents,and may be applicable for cancer Platelet-derived growth factor subunitB (isoform 1) (SEQ ID NO: 258) Stromal Derived Factor-1 Stromal GrowthFactor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 alphaGeneSeq Accession Cancer Res. 48: 4266) Hematopoietic, immune disorders,cancer Y39995 WO9948528 P48061-2/SDF1_HUMAN Isoform Alpha of Stromalcell-derived factor 1 (isoform alpha) (SEQ ID NO: 259) Stromal DerivedFactor-1 Stromal Growth Factor Proliferation assay using NR6R-3T3 cells(Rizzino 1988 beta GeneSeq Accession Cancer Res. 48: 4266)Hematopoietic, immune disorders, cancer R75420 CA2117953P48061/SDF1_HUMAN Stromal cell-derived factor 1 (isoform beta) (SEQ IDNO: 260) Tarc GeneSeq Accession Chemotactic for T lymphocytes. May playa role in T-cell development. Thought to W14917 WO9711969 bind CCR8 andCCR4 Chemotactic leukocyte migration assays are known in the art,Q92583/CCL17_HUMAN for example: J. Immunol. Methods 33 ((1980))Antiinflammatory. Immune disorders, C-C motif chemokine 17 cancer (SEQID NO: 261) Prolactin GeneSeq Prolactin is involved in immune cellproliferation and apoptosis. Immune coil Accession R78691 proliferationand suppression of apoptosis by prolactin can be assayed by methodsWO9521625 well-known in the art, for example, Buckley, AR and BuckleyDJ, Ann N Y Acad Sci P01236/PRL_HUMAN 2000; 917: 522-33, and within.Reproductive system disorders, cancer. Prolactin (SEQ ID NO: 262)Prolactin2 GeneSeq Prolactin is involved in immune cell proliferationand apoptosis. Immune coil Accession Y31764 U.S. proliferation andsuppression of apoptosis by prolactin can be assayed by methods Pat. No.5,955,346 well-known in the art, for example, Buckley, AR and BuckleyDJ, Ann N Y Acad Sci 2000; 917: 522-33, and within. Reproductive systemdisorders, cancer. Follicle stimulating FSH stimulates secretion ofinterleukin-1 by cells isolated from women in the follicular hormoneAlpha subunit phase FSH activities can be determined using assays knownin the art; J Gend Specif GeneSeq Accession Med 1999 Nov-Dec; 2(6):30-4; Mol Cell Endocrinol. 1997 Nov. 15; 134(2): 109-18. Y54160 EP974359Reproductive system disorders, cancer. P01215/GLHA_HUMAN Glycoproteinhormones alpha chain (SEQ ID NO: 263) Follicle stimulating FSHstimulates secretion of interleukin-1 by cells isolated from women inthe follicular hormone Beta subunit phase FSH activities can bedetermined using assays known in the art; J Gend Specif GeneSeqAccession Med 1999 Nov-Dec; 2(6): 30-4; Mol Cell Endocrinol. 1997 Nov.15; 134(2): 109-18. Y54161 EP974359 Reproductive system disorders,cancer. P01225/FSHB_HUMAN Follitropin subunit beta (SEQ ID NO: 264)Substance P (tachykinin) Substance P is associated withimmunoregulation. Immuneregulation and bone GeneSeq Accession marrow,cell proliferation by substance P can be assayed by methods well-knownin B23027 WO0054053 the art, for example, Lai et al. Proc Natl Acad SciUSA 2001 Mar. 27; 98(7): 3970-5; (SEQ ID NO: 385) Jallat-Daloz et al.Allergy Asthma Proc 2001 Jan-Feb; 22(1): 17-23; Kahler et al. Exp LungRes 2001 Jan-Feb; 27(1): 25-46; and Adamus MA and Dabrowski ZJ. J CellBiochem 2001; 81(3)499-506. diabetes mellitus, hypertension, cancerOxytocin (Neurophysin I) Oxytocin is involved in the induction ofprostaglandin (E2) release as well as an GeneSeq Accession increasedamount of calcium release by smooth muscle cells. Oxytocin and B24085and B24086 prostaglandin E(2) release and Ocytocin (Ca2+) increase canbe assayed by WO0053755 methods well-known in the art, for example,Pavan et al., AM J Obset Gynecol 2000 P01178/NEU1_HUMAN Jul; 183(1):76-82 and Holda et al., Cell Calcium 1996 Jul; 20(1): 43 51.inflammatory Oxytocin-neurophysin 1 disorders immunologic disorders,cancer (SEQ ID NO: 265) Vasopressin (Neurophysin Vasopressinis believedto have a direct antidiuretic action on the kidney, and it is II)GeneSeq Accession thought to cause vasoconstriction of the peripheralvessels. Vasopressin activity can B24085 and B24086 be determined usingassays known in the art, for example, Endocr Regul 1996 Mar; WO005375530(I): 13-17. inflammatory disorders immunologic disorders, cancerP01185/NEU2_HUMAN Vasopressin-neurophysin 2-copeptin (SEQ ID NO: 266)IL-1 GeneSeq Accession Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, P60326 EP165654 monocytes, andmacrophages. Known functions include stimulating proliferation of IL-1alpha immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis P01583|IL1A_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-1 alpha can be determined using assays known in the art:Matthews et al., in Lymphokines (SEQ ID NO: 269) and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, IL-1beta D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1,14-20. P01584|IL1B_HUMAN inflammatory disorders immunologic disorders,cancer Interleukin-1 beta (SEQ ID NO: 267) IL-1 mature GeneSeqInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, Accession R14855 monocytes, and macrophages. Knownfunctions include stimulating proliferation of EP456332 immune cells(e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis (mature truncated form of neutrophils and T lymphocytes,and/or inhibition of interferons. Interleukin activity wherein theprecursor is can be determined using assays known in the art: Matthewset al., in Lymphokines cleaved between amino and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, acids116-117) D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989)Cytokine 1, 14-20. (SEQ ID NO: 386) inflammatory disorders immunologicdisorders, cancer IL-1 beta GeneSeq Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, Accession Y08322monocytes, and macrophages. Known functions include stimulatingproliferation of WO9922763 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis P01584|IL1B_HUMAN ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Interleukin-1 beta can be determined using assaysknown in the art: Matthews et al., in Lymphokines (SEQ ID NO: 267) andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989)Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancerIL-3 variants GeneSeq Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, Accession P80382, monocytes, andmacrophages. Known functions include stimulating proliferation ofP80383, P80384, and immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis P80381 WO8806161 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Variants of wildtype IL-3 can be determined usingassays known in the art: Matthews et al., in Lymphokines which has thesequence: and Interferens: A Practical Approach, Clemens et al., eds,IRL Press, Washington, P08700|IL3_HUMAN D.C. 1987, pp. 221-225; andKitamura et al (1989) J Cell Physiol. 140 323-334. Interleukin-3inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 449)IL-4 GeneSeq Accession Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, P70615 WO8702990 monocytes, andmacrophages. Known functions include stimulating proliferation ofP05112/IL4_HUMAN immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis Interleukin-4 of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity (isoform 1) can be determined using assays known in the art:Matthews et al., in Lymphokines (SEQ ID NO: 268) and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132,287-295. inflammatory disorders immunologic disorders, cancer IL-4muteins GeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession W52151 monocytes, and macrophages.Known functions include stimulating proliferation of W52152 W52153W52154 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis W52155 W52156 W52157 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityW52158 W52159 W52160 can be determined using assays known in the art:Matthews et al., in Lymphokines W52161 W52162 W52163 and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, W52164and W52165 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) JImmunol Methods 132, WO9747744 287-295. inflammatory disordersimmunologic disorders, cancer Variants of wildtype IL-4 which has thesequence: P05112/IL4_HUMAN Interleukin-4 (isoform 1) (SEQ ID NO: 268)IL-1 alpha GeneSeq Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, Accession P90108 monocytes, andmacrophages. Known functions include stimulating proliferation ofEP324447 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis P01583|IL1A_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-1 alpha can be determined using assays known in the art:Matthews et al., in Lymphokines (SEQ ID NO: 269) and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20.inflammatory disorders immunologic disorders, cancer IL-3 variantsGeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession R38561, monocytes, andmacrophages. Known functions include stimulating proliferation ofR38562, R38563, R38564, immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis R38565, R38566, R38567, ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity R38568, R38569, R38570, can be determined usingassays known in the art: Matthews et al., in Lymphokines R38571, andR38572 and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, WO9307171 D.C. 1987, pp. 221-225; and Aarden et al(1987) Eur. J. Immunol 17, 1411-16. Variants of wildtype IL-3inflammatory disorders immunologic disorders, cancer which has thesequence: P08700|IL3_HUMAN Interleukin-3 (SEQ ID NO: 449) IL-6 GeneSeqAccession Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, R45717 and R45718 monocytes, andmacrophages. Known functions include stimulating proliferation ofWO9402512 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis P05231/IL6_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-6 can be determined using assays known in the art: Matthewset al., in Lymphokines (SEQ ID NO: 270) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16.inflammatory disorders immunologic disorders, cancer. Obesity. MetabolicDisease. Diabetes. IL-13 GeneSeq Accession Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, R48624 WO9404680monocytes, and macrophages. Known functions include stimulatingproliferation of P35225/IL13_HUMAN immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis Interleukin-13 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity (SEQ ID NO: 271) can be determined using assaysknown in the art: Matthews et al., in Lymphokines and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Boutelier et al (1995) J. Immunol. Methods 181,29. inflammatory disorders immunologic disorders, cancer IL-4 muteinGeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession R47182 monocytes, and macrophages.Known functions include stimulating proliferation of DE4137333 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis Variants of wildtype IL-4 of neutrophils and T lymphocytes,and/or inhibition of interferons. Interleukin activity which has thesequence: can be determined using assays known in the art: Matthews etal., in Lymphokines P05112/IL4_HUMAN and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, Interleukin-4 D.C.1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132,(isoform 1) 287-295. inflammatory disorders immunologic disorders,cancer (SEQ ID NO: 268) IL-4 mutein Y124X Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, GeneSeq Accessionmonocytes, and macrophages. Known functions include stimulatingproliferation of R47183 DE4137333 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis Variants of wildtypeIL-4 of neutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity which has the sequence: can be determined usingassays known in the art: Matthews et al., in LymphokinesP05112/IL4_HUMAN and Interferens: A Practical Approach, Clemens et al.,eds, IRL Press, Washington, Interleukin-4 D.C. 1987, pp. 221-225; andSiegel & Mostowski (1990) J Immunol Methods 132, (isoform 1) 287-295.inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 268))IL-4 mutein Y124G Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, GeneSeq Accession monocytes, andmacrophages. Known functions include stimulating proliferation of R47184DE4137333 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis Variants of wildtype IL-4 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activitywhich has the sequence: can be determined using assays known in the art:Matthews et al., in Lymphokines P05112/IL4_HUMAN and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington,Interleukin-4 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) JImmunol Methods 132, (isoform 1) 287-295. inflammatory disordersimmunologic disorders, cancer (SEQ ID NO: 268) Human Interleukin-10Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, (precursor) GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession R41664 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9317698 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity P22301/IL10_HUMAN can be determinedusing assays known in the art: Matthews et al., in LymphokinesInterleukin-10 and Interferens: A Practical Approach, Clemens et al.,eds, IRL Press, Washington, (precursor form is D.C. 1987, pp. 221-225;and Thompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. processedinto a truncated inflammatory disorders immunologic disorders, cancermature form) (SEQ ID NO: 272) Human Interleukin-10 Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, GeneSeqAccession monocytes, and macrophages. Known functions includestimulating proliferation of R42642 WO9318783-A immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ IDNO: 3 of of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity WO9318783-A can be determined usingassays known in the art: Matthews et al., in Lymphokines (mature IL-10)and Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (SEQ ID NO: 273) D.C. 1987, pp. 221-225; and Thompson-Snipeset al (1991) J. Exp. Med. 173, 507-510. inflammatory disordersimmunologic disorders, cancer Human interleukin-1 beta Interleukins area group of multi-functional cytokines synthesized by lymphocytes,precursor. GeneSeq monocytes, and macrophages. Known functions includestimulating proliferation of Accession R42447 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisEP569042 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity P01584/IL1B_HUMAN can be determinedusing assays known in the art: Matthews et al., in LymphokinesInterleukin-1 beta and Interferens: A Practical Approach, Clemens etal., eds, IRL Press, Washington, (SEQ ID NO: 274) D.C. 1987, pp.221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. inflammatorydisorders immunologic disorders, cancer Interleukin-1alpha Interleukinsare a group of multi-functional cytokines synthesized by lymphocytes,GeneSeq Accession monocytes, and macrophages. Known functions includestimulating proliferation of R45364 EP578278 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisP01583|IL1A_HUMAN of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Interleukin-1 alpha can be determinedusing assays known in the art: Matthews et al., in Lymphokines (SEQ IDNO: 269) and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, D.C. 1987, pp. 221-225. inflammatory disordersimmunologic disorders, cancer Human interleukin-3 Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, variantGeneSeq monocytes, and macrophages. Known functions include stimulatingproliferation of Accession R22814 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis JP04063595 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Variants of wildtype IL-3 can be determined usingassays known in the art: Matthews et al., in Lymphokines which has thesequence: and Interferens: A Practical Approach, Clemens et al., eds,IRL Press, Washington, P08700|IL3_HUMAN D.C. 1987, pp. 221-225; Kitamuraet al (1989) J Cell Physiol. 140 323-334. Interleukin-3 inflammatorydisorders immunologic disorders, cancer (SEQ ID NO: 449) IL-1i fragmentsGeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession R35484 and monocytes, andmacrophages. Known functions include stimulating proliferation of R35485EP541920 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity can be determined usingassays known in the art: Matthews et al., in Lymphokines andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989)Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancerIL-1 inhibitor (IL-li) Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, GeneSeq Accession monocytes, andmacrophages. Known functions include stimulating proliferation of R35486and R35484 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis EPS541920 of neutrophils and T lymphocytes,and/or inhibition of interferons. Interleukin activity can be determinedusing assays known in the art: Matthews et al., in Lymphokines andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989)inflammatory disorders immunologic disorders, cancer ICE 22 kD subunit.Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of R33780 EP533350 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis SEQ ID NO: 16 of of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity EP533350 can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 450) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225.inflammatory disorders immunologic disorders, cancer ICE 20 kD subunit.Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of R33781 EP533350 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis SEQ ID NO: 17 of of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity EP533350 can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 451) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225.inflammatory disorders immunologic disorders, cancer ICE 10 kD subunitInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of R33782 EP533350 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis SEQ ID NO: 18 of of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity EP533350 can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 452) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225.inflammatory disorders immunologic disorders, cancer HumanInterleukin-10 Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, (precursor) GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession R41664 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis WO9317698 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityP22301/IL10_HUMAN can be determined using assays known in the art:Matthews et al., in Lymphokines Interleukin-10 and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington,(precursor form is D.C. 1987, pp. 221-225; and Thompson-Snipes et al(1991) J. Exp. Med. 173, 507-510. processed into a truncatedinflammatory disorders immunologic disorders, cancer mature form) (SEQID NO: 272) Human Interleukin-10 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, GeneSeq Accessionmonocytes, and macrophages. Known functions include stimulatingproliferation of R42642 WO9318783 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 3 of ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity WO9318783-A can be determined using assays known inthe art: Matthews et al., in Lymphokines (mature IL-10) and Interferens:A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQID NO: 273) D.C. 1987, pp. 221-225; and Thompson-Snipes et al (1991) J.Exp. Med. 173, 507-510. inflammatory disorders immunologic disorders,cancer Human Interleukin-1 beta Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, precursor GeneSeqmonocytes, and macrophages. Known functions include stimulatingproliferation of Accession R42447 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis EP569042 of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity P01584/IL1B_HUMAN can be determined using assays known in theart: Matthews et al., in Lymphokines Interleukin-1 beta and Interferens:A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQID NO: 274) D.C. 1987, pp. 221-225; Kitamura et al (1989) J CellPhysiol. 140 323-334. inflammatory disorders immunologic disorders,cancer Human interleukin-6 Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, GeneSeq Accession monocytes, andmacrophages. Known functions include stimulating proliferation of R49041WO9403492 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis P05231/IL6_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-6 can be determined using assays known in the art: Matthewset al., in Lymphokines (SEQ ID NO: 270) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16.inflammatory disorders immunologic disorders, cancer Mutant Interleukin6 Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, S176R GeneSeq monocytes, and macrophages. Known functionsinclude stimulating proliferation of Accession R54990 immune cells(e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9411402 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity S176R variant of wildtype can bedetermined using assays known in the art: Matthews et al., inLymphokines IL-6 which has the and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, sequence: D.C. 1987, pp.221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16.P05231/IL6_HUMAN inflammatory disorders immunologic disorders, cancerInterleukin-6 (SEQ ID NO: 270) Interleukin 6 GeneSeq Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes,Accession R55256 monocytes, and macrophages. Known functions includestimulating proliferation of JP06145063 immune cells (e.g., T helpercells, B cells, eosinophils, and lymphocytes), chemotaxisP05231/IL6_HUMAN of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Interleukin-6 can be determined usingassays known in the art: Matthews et al., in Lymphokines (SEQ ID NO:270) and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur.J. Immunol 17, 1411-16. inflammatory disorders immunologic disorders,cancer Interleukin 8 (IL-8) Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, receptor GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession R53932 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis JP06100595 of neutrophils andT lymphocytes, and/or inhibition of interferons. Interleukin activityGenBank: AAA59159.1 can be determined using assays known in the art:Matthews et al., in Lymphokines (SEQ ID NO: 275) and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80. SolubleIL-8 receptor polypeptides may be useful for inhibiting interleukinactivities. Human interleukin-7 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, GeneSeq Accessionmonocytes, and macrophages. Known functions include stimulatingproliferation of R59919 U.S. Pat. No. immune cells (e.g., T helpercells, B cells, eosinophils, and lymphocytes), chemotaxis 5,328,988 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity P13232/IL7_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Interleukin-7 andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (isoform 1) D.C. 1987, pp. 221-225; and Park et al (1990) J.Exp. Med. 171, 1073-79. (SEQ ID NO: 276) inflammatory disordersimmunologic disorders, cancer IL-3 containing fusion Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, protein.GeneSeq monocytes, and macrophages. Known functions include stimulatingproliferation of Accession R79342 and immune cells (e.g., T helpercells, B cells, eosinophils, and lymphocytes), chemotaxis R79344WO9521254 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Fusions of wildtype IL-3 can bedetermined using assays known in the art: Matthews et al., inLymphokines which has the sequence: and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, P08700|IL3_HUMAND.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140323-334. Interleukin-3 inflammatory disorders immunologic disorders,cancer (SEQ ID NO: 449) IL-3 mutant proteins Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, GeneSeq Accessionmonocytes, and macrophages. Known functions include stimulatingproliferation of R79254, R79255, R79256, immune cells (e.g., T helpercells, B cells, eosinophils, and lymphocytes), chemotaxis R79257,R79258, R79259, of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity R79260, R79261, R79262, can bedetermined using assays known in the art: Matthews et al., inLymphokines R79263, R79264, R79265, and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, R79266, R79267,R79268, D.C. 1987, pp. 221-225; and Giri et al (1994) EMBO J. 132822-2830. inflammatory R79269, R79270, R79271, disorders immunologicdisorders, cancer R79272, R79273, R79274, R79275, R79276, R79277,R79278, R79279, R79280, R79281, R79282, R79283, R79284, and R79285ZA9402636 Variants of wildtype IL-3 which has the sequence:P08700|IL3_HUMAN Interleukin-3 (SEQ ID NO: 449) IL-12 p40 subunit.Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of R63018 AU9466072 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis P2946/|IL12B_HUMAN of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity Interleukin-12 subunitbeta can be determined using assays known in the art: Matthews et al.,in Lymphokines (SEQ ID NO: 277) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225.inflammatory disorders immunologic disorders, cancer AGF GeneSeqAccession Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, R64240 WO9429344 monocytes, and macrophages.Known functions include stimulating proliferation of Q8NI99/ANGL6_HUMANimmune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis Angiopoietin-related of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityprotein 6 can be determined using assays known in the art: Matthews etal., in Lymphokines (SEQ ID NO: 278) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225. inflammatory disorders immunologic disorders, cancer Humaninterlaukin-12 40 kD Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, subunit GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession R79187 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis WO9519786 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityP2946/|IL12B_HUMAN can be determined using assays known in the art:Matthews et al., in Lymphokines Interleukin-12 subunit beta andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (SEQ ID NO: 277) D.C. 1987, pp. 221-225; and Hori et al(1987), Blood 70, 1069-1078. inflammatory disorders immunologicdisorders, cancer Human interleukin-15 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, receptor fromclone P1 monocytes, and macrophages. Known functions include stimulatingproliferation of GeneSeq Accession immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis R90843 WO9530695 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Q13261|I15RA_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Interleukin-15receptor and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, subunit alpha D.C. 1987, pp. 221-225; and Giri et al(1994) EMBO J. 13 2822-2830. Soluble IL-8 Isoform 1) receptorpolypeptides may be useful for inhibiting interleukin activities.Obesity. (SEQ ID NO: 453) Metabolic Disease. Diabetes. Enhancingsecretion and stability of Interleukin 15 Human interleukin-7Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of R92796 WO9604306 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis P13232/IL7_HUMAN of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity Interleukin-7 can bedetermined using assays known in the art: Matthews et al., inLymphokines (isoform 1) and Interferens: A Practical Approach, Clemenset al., eds, IRL Press, Washington, (SEQ ID NO: 276) D.C. 1987, pp.221-225; and Park et al (1990) J. Exp. Med. 171, 1073-79. inflammatorydisorders immunologic disorders, cancer interleukin-9 GeneSeqInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, Accession R92797 monocytes, and macrophages. Knownfunctions include stimulating proliferation of WO9604306 immune cells(e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis P15248/IL9_HUMAN of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity Interleukin-9 can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 279) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; andYang et al (1989) Blood 74, 1880-84. inflammatory disorders immunologicdisorders, cancer interleukin-3 GeneSeq Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, Accession R92801monocytes, and macrophages. Known functions include stimulatingproliferation of WO9604306 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis P08700|IL3_HUMAN ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Interleukin-3 can be determined using assays knownin the art: Matthews et al., in Lymphokines (SEQ ID NO: 280) andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; Kitamura et al (1989) J CellPhysiol. 140 323-334. inflammatory disorders immunologic disorders,cancer Human interleukin-5 Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, GeneSeq Accession monocytes, andmacrophages. Known functions include stimulating proliferation of R92802WO9604306 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis P05113/IL5_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-5 can be determined using assays known in the art: Matthewset al., in Lymphokines (SEQ ID NO: 281) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. inflammatorydisorders immunologic disorders, cancer Recombinant interleukin-Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, 16 GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of W33373 DE19617202 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis Q14005/IL16_HUMAN of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity Pro-interleukin-16 canbe determined using assays known in the art: Matthews et al., inLymphokines (isoform 1) and Interferens: A Practical Approach, Clemenset al., eds, IRL Press, Washington, (SEQ ID NO: 282) D.C. 1987, pp.221-225; and Lim et al (1996) J. Immunol. 156, 2566-70. inflammatorydisorders immunologic disorders, cancer Human IL-16 protein Interleukinsare a group of multi-functional cytokines synthesized by lymphocytes,GeneSeq Accession monocytes, and macrophages. Known functions includestimulating proliferation of W33234 DE19617202 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisQ14005/IL16_HUMAN of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Pro-interleukin-16 can be determinedusing assays known in the art: Matthews et al., in Lymphokines(isoform 1) and Interferens: A Practical Approach, Clemens et al., eds,IRL Press, Washington, (SEQ ID NO: 282) D.C. 1987, pp. 221-225; and Limet al (1996) J. Immunol. 156, 2566-70. inflammatory disordersimmunologic disorders, cancer Thrl 17 human interleukin Interleukins area group of multi-functional cytokines synthesized by lymphocytes, 9GeneSeq Accession monocytes, and macrophages. Known functions includestimulating proliferation of W27521 WO9708321 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisP15248|IL9_HUMAN of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Interleukin-9 can be determined usingassays known in the art: Matthews et al., in Lymphokines (SEQ ID NO:387) and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, D.C. 1987, pp. 221-225. inflammatory disordersimmunologic disorders, cancer Metl 17 human interleukin Interleukins area group of multi-functional cytokines synthesized by lymphocytes, 9GeneSeq Accession monocytes, and macrophages. Known functions includestimulating proliferation of W27522 WO9708321 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis (SEQ IDNO: 388) of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity can be determined using assays knownin the art: Matthews et al., in Lymphokines and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory disordersimmunologic disorders, cancer Human intracellular IL-1 Interleukins area group of multi-functional cytokines synthesized by lymphocytes,receptor antagonist. monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis W77158EP864585 (e.g. of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity SEQ ID NOs: 12 to 19, or can bedetermined using assays known in the art: Matthews et al., inLymphokines 22 to 25 of this and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, publication). D.C. 1987, pp.221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. inflammatorydisorders immunologic disorders, cancer Human interleukin-18Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, protein (IL-18) GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession W77158 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis EP864585 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity Q14116/IL18_HUMAN can be determinedusing assays known in the art: Matthews et al., in LymphokinesInterleukin-18 and Interferens: A Practical Approach, Clemens et al.,eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; andUSHIO et al (1996) J. Immunol. 156, 4274-79. (SEQ ID NO: 283)inflammatory disorders immunologic disorders, cancer Humaninterleukin-18 Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, GeneSeq Accession monocytes, andmacrophages. Known functions include stimulating proliferation of W77077EP861663 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis Q14116/IL18_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-18 can be determined using assays known in the art: Matthewset al., in Lymphokines (isoform 1) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 283)D.C. 1987, pp. 221-225; and USHIO et al (1996) J. Immunol. 156, 4274-79.inflammatory disorders immunologic disorders, cancer Human interleukin18 Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, derivatives GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accessions W77083, immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis W77084, W77085, of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity W77086, W77087, can bedetermined using assays known in the art: Matthews et al., inLymphokines W77088, and W77089 and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, EP861663 D.C. 1987, pp.221-225; and Ushio et al (1996) J. Immunol, 156, 4274-79. Variants ofwildtype IL18 inflammatory disorders immunologic disorders, cancer whichis provided as: Q14116/IL18_HUMAN Interleukin-18 (isoform 1) (SEQ ID NO:283) Interleukin-9 (IL-9) mature Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, protein (Thr117version). monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis W68158WO9827997 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity FIG. 2 of WO9827997 can be determinedusing assays known in the art: Matthews et al., in Lymphokines (SEQ IDNO: 389) and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, D.C. 1987, pp. 221-225; and Yang et al (1989) Blood74, 1880-84. inflammatory disorders immunologic disorders, cancer IL-9mature protein variant Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, (Met117 version) GenSeq monocytes,and macrophages. Known functions include stimulating proliferation ofAccession W68157 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis WO9827997 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activity FIG.3 of WO9827997 can be determined using assays known in the art: Matthewset al., in Lymphokines (SEQ ID NO: 390) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory disordersimmunologic disorders, cancer Human IL-9 receptor Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, proteinvariant #3. monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis W64058WO9824904 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Wildtype IL-9R is provided can bedetermined using assays known in the art: Matthews et al., inLymphokines as: and Interferens: A Practical Approach, Clemens et al.,eds, IRL Press, Washington, Q01113/IL9R_HUMAN D.C. 1987, pp. 221-225;and Yang et al (1989) Blood 74, 1880-84. inflammatory Interleukin-9receptor disorders immunologic disorders, cancer (isoform 1) (SEQ ID NO:303) Human IL-9 receptor Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, protein variant fragmentmonocytes, and macrophages. Known functions include stimulatingproliferation of GenSeq Accession immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis W64060 WO9824904 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Wildtype IL-9R is provided can be determined usingassays known in the art: Matthews et al., in Lymphokines as: andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, Q01113/IL9R_HUMAN D.C. 1987, pp. 221-225; and Yang et al(1989) Blood 74, 1880-84. Soluble IL-9 Interleukin-9 receptor receptorpolypeptides may be useful for inhibiting interleukin activities.(isoform 1) (SEQ ID NO: 303) Human IL-9 receptor Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, proteinvariant #3. monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis W64061WO9824904 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Wildtype IL-9R is provided can bedetermined using assays known in the art: Matthews et al., inLymphokines as: and Interferens: A Practical Approach, Clemens et al.,eds, IRL Press, Washington, Q01113/IL9R_HUMAN D.C. 1987, pp. 221-225;and Yang et al (1989) Blood 74, 1880-84. Soluble IL-9 Interleukin-9receptor receptor polypeptides may be useful for inhibiting interleukinactivities. (isoform 1) (SEQ ID NO: 303) Human Interleukin-12 p40Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, protein GeneSeq monocytes, and macrophages. Known functionsinclude stimulating proliferation of Accession W51311 immune cells(e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9817689 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity P2946/|IL12B_HUMAN can bedetermined using assays known in the art: Matthews et al., inLymphokines Interleukin-12 subunit beta and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 277)D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078.inflammatory disorders immunologic disorders, cancer HumanInterleukin-12 p35 Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, protein GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession W51312 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis WO9817689 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityP29459/IL12A_HUMAN can be determined using assays known in the art:Matthews et al., in Lymphokines Interleukin-12 subunit and Interferens:A Practical Approach, Clemens et al., eds, IRL Press, Washington, alphaD.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078.inflammatory (SEQ ID NO: 284) disorders immunologic disorders, cancerHuman protein with IL-16 Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, activity GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession W63753 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis DE19649233- of neutrophils andT lymphocytes, and/or inhibition of interferons. Interleukin activitycan be determined using assays known in the art: Matthews et al., inLymphokines and Interferens: A Practical Approach, Clemens et al., eds,IRL Press, Washington, D.C. 1987, pp. 221-225; and Lim et al (1996) J.Immunol. 156, 2566-70. inflammatory disorders immunologic disorders,cancer Human protein with IL-16 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, activity GeneSeqmonocytes, and macrophages. Known functions include stimulatingproliferation of Accession W59425 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis DE19649233- ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity can be determined using assays known in the art:Matthews et al., in Lymphokines and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; andLim et al (1996) J. Immunol. 156, 2566-70. inflammatory disordersimmunologic disorders, cancer Human interleukin-15 Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, GeneSeqAccession monocytes, and macrophages. Known functions includestimulating proliferation of W53878 U.S. Pat. No. immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis5,747,024 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity P40933/IL15_HUMAN can be determinedusing assays known in the art: Matthews et al., in LymphokinesInterleukin-15 and Interferens: A Practical Approach, Clemens et al.,eds, IRL Press, Washington, (isoform IL15-S48AA) D.C. 1987, pp. 221-225;and Giri et al (1994) EMBO J. 13 2822-2830. inflammatory (SEQ ID NO:285) disorders immunologic disorders, cancer. Obesity. MetabolicDisease. Diabetes. Human wild-type Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, interleukin-4(hIL-4) monocytes, and macrophages. Known functions include stimulatingproliferation of protein GeneSeq immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis Accession W52149 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity WO9747744 can be determined using assays known inthe art: Matthews et al., in Lymphokines P05112/IL4_HUMAN andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, Interleukin-4 D.C. 1987, pp. 221-225; and Siegel & Mostowski(1990) J Immunol Methods 132, (isoform 1) 287-295. inflammatorydisorders immunologic disorders, cancer (SEQ ID NO: 286) interleukin-4muteins Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, GeneSeq Accessions monocytes, andmacrophages. Known functions include stimulating proliferation ofW52150, W52151, immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis W52153, W52154, of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity W52155, W52156, can be determined using assays known in theart: Matthews et al., in Lymphokines W52157, W52158, and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, W52159,W52160, D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J ImmunolMethods 132, W52161, W52162, 287-295. inflammatory disorders immunologicdisorders, cancer W52163, W52164, W52165, W52166, and W52167 WO9747744Variants of wildtype IL-4 which has the sequence: P05112/IL4_HUMANInterleukin-4 (isoform 1) (SEQ ID NO: 268) Human interleukin 1 deltaInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of Y28408 WO9935268 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis SEQ ID NO: 4 of of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity WO9935268 can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 287) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; andOrencole & Dinarello (1989) Cytokine 1, 14-20. inflammatory disordersimmunologic disorders, cancer Human interleukin-1 Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, receptorantagonist beta monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis Y24395WO9935268 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity can be determined using assays knownin the art: Matthews et al., in Lymphokines and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. inflammatorydisorders immunologic disorders, cancer Human EDIRF II proteinInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, sequence GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession Y22199 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9932632 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity SEQ ID NO: 6 of can be determinedusing assays known in the art: Matthews et al., in Lymphokines WO9932632and Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (SEQ ID NO: 391) D.C. 1987, pp. 221-225. inflammatorydisorders immunologic disorders, cancer Human EDIRF I proteinInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, sequence GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession Y22197 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9932632 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity SEQ ID NO: 2 of can be determinedusing assays known in the art: Matthews et al., in Lymphokines WO9932632and Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (SEQ ID NO: 392) D.C. 1987, pp. 221-225. inflammatorydisorders immunologic disorders, cancer Human IL-1RD10 proteinInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, sequence GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession Y14131 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9919480 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity SEQ ID NO: 20 of can be determinedusing assays known in the art: Matthews et al., in Lymphokines WO9919480and Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989)Cytokine 1, 14-20. Soluble IL-1RD10 receptor polypeptides may be usefulfor inhibiting interleukin activites. Human IL-1RD9 Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, GeneSeqAccession monocytes, and macrophages. Known functions includestimulating proliferation of Y14122 WO9919480 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ IDNOS: 6, 8, 10 of of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity WO9919480 can be determined usingassays known in the art: Matthews et al., in Lymphokines andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989)Cytokine 1, 14-20. Soluble IL-1RD10 receptor polypeptides may be usefulfor inhibiting interleukin activites. Human DNAX interleukin-Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, 40 GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of Y09196 WO9919491 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis SEQ ID NO: 2 or 4 of of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity WO9919491 can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 454) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225.inflammatory disorders immunologic disorders, cancer (DIL-40)alternative Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, sequence GeneSeq monocytes, and macrophages.Known functions include stimulating proliferation of Accession Y09197immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis WO9919491 of neutrophils and T lymphocytes,and/or inhibition of interferons. Interleukin activity SEQ ID NO: 4 ofcan be determined using assays known in the art: Matthews et al., inLymphokines WO9919491 and Interferens: A Practical Approach, Clemens etal., eds, IRL Press, Washington, (SEQ ID NO: 455) D.C. 1987, pp.221-225. inflammatory disorders immunologic disorders, cancer IL-11GeneSeq Accession Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, R50176 WO9405318 monocytes, and macrophages.Known functions include stimulating proliferation of P2080/|IL11_HUMANimmune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis Interleukin-11 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activity(isoform 1) can be determined using assays known in the art: Matthews etal., in Lymphokines (SEQ ID NO: 288) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Lu et al (1994) J immunol. Methods 173, 19. inflammatorydisorders immunologic disorders, cancer Human adipogenesis Interleukinsare a group of multi-functional cytokines synthesized by lymphocytes,inhibitory factor GeneSeq monocytes, and macrophages. Known functionsinclude stimulating proliferation of Accession R43260 immune cells(e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis EP566410 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity (also known as IL-11) can bedetermined using assays known in the art: Matthews et al., inLymphokines P2080/|IL11_HUMAN and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, Interleukin-11 D.C. 1987,pp. 221-225. inflammatory disorders immunologic disorders, cancer(isoform 1) (SEQ ID NO: 288) IL-11 GeneSeq Accession Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, W02202JP08127539 monocytes, and macrophages. Known functions includestimulating proliferation of P2080/|IL11_HUMAN immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisInterleukin-11 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity (isoform 1) can be determined usingassays known in the art: Matthews et al., in Lymphokines (SEQ ID NO:288) and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, D.C. 1987, pp. 221-225; and Lu et al (1994) Jimmunol. Methods 173, 19. inflammatory disorders immunologic disorders,cancer IL-14 GeneSeq Accession Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, R55800 WO9416074monocytes, and macrophages. Known functions include stimulatingproliferation of P40222/TXLNA_HUMAN immune cells (e.g., T helper cells,B cells, eosinophils, and lymphocytes), chemotaxis Alpha-taxilin ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity (SEQ ID NO: 289) can be determined using assaysknown in the art: Matthews et al., in Lymphokines and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Ambrus et al (1993) PNAS 90, 63330-34.inflammatory disorders immunologic disorders, cancer IL-17 receptorGeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession B03807 U.S. monocytes, andmacrophages. Known functions include stimulating proliferation of Pat.No. 6,072,033 immune cells (e.g., T helper cells, B cells, eosinophils,and lymphocytes), chemotaxis Q96F46/I17RA_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-17 receptor A can be determined using assays known in theart: Matthews et al., in Lymphokines (SEQ ID NO: 290) and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86.Soluble IL- 17 receptor polypeptides may be useful for inhibitinginterleukin activites. IL-17 GeneSeq Accession Interleukins are a groupof multi-functional cytokines synthesized by lymphocytes, R76573WO9518826 monocytes, and macrophages. Known functions includestimulating proliferation of Q16552/IL17_HUMAN immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisInterleukin-17A of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity (SEQ ID NO: 291) can be determinedusing assays known in the art: Matthews et al., in Lymphokines andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol.155, 5483-86. inflammatory disorders immunologic disorders, cancerCTLA-8 GeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession W13651 monocytes, and macrophages.Known functions include stimulating proliferation of WO9704097 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis (also known as IL-17) of neutrophils and T lymphocytes,and/or inhibition of interferons. Interleukin activity Q16552/IL17_HUMANcan be determined using assays known in the art: Matthews et al., inLymphokines Interleukin-17A and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 291) D.C. 1987,pp. 221-225. inflammatory disorders immunologic disorders, cancer IL-19GeneSeq Accession Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, W37935 WO9808870 monocytes, and macrophages.Known functions include stimulating proliferation of Q9UHD0|IL19_HUMANimmune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis Interleukin-19 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activity(isoform 1) can be determined using assays known in the art: Matthews etal., in Lymphokines (SEQ ID NO: 292) and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp.221-225; and Gallagher et al (2000) Genes Immun. 1, 442-50. inflammatorydisorders immunologic disorders, cancer IL-21 (TIF) GeneSeq Interleukinsare a group of multi-functional cytokines synthesized by lymphocytes,Accession Y92879 monocytes, and macrophages. Known functions includestimulating proliferation of WO0024758 immune cells (e.g., T helpercells, B cells, eosinophils, and lymphocytes), chemotaxisQ9HBE4/IL21_HUMAN of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Interleukin-21 can be determined usingassays known in the art: Matthews et al., in Lymphokines (isoform 1) andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (SEQ ID NO: 293) D.C. 1987, pp. 221-225; and Parrish-Novaket al (2000) Nature 408, 57-63. inflammatory disorders immunologicdisorders, cancer IL-8 receptor GeneSeq Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, Accession R33420monocytes, and macrophages. Known functions include stimulatingproliferation of WO9306229 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis IL-8RA of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityP25024/CXCR1_HUMAN can be determined using assays known in the art:Matthews et al., in Lymphokines C—X—C chemokine receptor andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, type 1 D.C. 1987, pp. 221-225; and Holmes et al (1991)Science 253, 1278-80. Soluble IL-8 (SEQ ID NO: 294) receptorpolypeptides may be useful for inhibiting interleukin activities. IL-8RBP25025/CXCR2_HUMAN C—X—C chemokine receptor type 2 (SEQ ID NO: 295)Human type II interleukin- Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, 1 receptor GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession R85480 U.S. immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis Pat. No. 5,464,937 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity P27930/IL1R2_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Interleukin-1 receptortype 2 and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, (SEQ ID NO: 296) D.C. 1987, pp. 221-225; and Orencole& Dinarello (1989) Cytokine 1, 14-20. Soluble type II interleukin-1receptor polypeptides may be useful for inhibiting interleukinactivities. Human interleukin-12 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, receptor GeneSeqmonocytes, and macrophages. Known functions include stimulatingproliferation of Accession R69632 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis EP638644 of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity IL-12 receptor B1 can be determined using assays known in theart: Matthews et al., in Lymphokines P42701|I12R1_HUMAN and Interferens:A Practical Approach, Clemens et al., eds, IRL Press, Washington,Interleukin-12 receptor D.C. 1987, pp. 221-225; and Hori et al (1987),Blood 70, 1069-1078. Soluble IL-12 subunit beta-1 receptor polypeptidesmay be useful for inhibiting interleukin activities. (isoform 1) (SEQ IDNO: 393) IL-12 receptor B2 Q99665|I12R2_HUMAN Interleukin-12 receptorsubunit beta-2 (isoform 1) (SEQ ID NO: 394) Interleukin 8 receptor BInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, GeneSeq Accession monocytes, and macrophages. Knownfunctions include stimulating proliferation of R80758 U.S. Pat. No.immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis 5,440,021 of neutrophils and T lymphocytes,and/or inhibition of interferons. Interleukin activity IL-8RB can bedetermined using assays known in the art: Matthews et al., inLymphokines P25025/CXCR2_HUMAN and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, C—X—C chemokine receptorD.C. 1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80.Soluble IL-8 type 2 receptor B polypeptides may be useful for inhibitinginterleukin activities. (SEQ ID NO: 295) Human IL-8 receptorInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, protein hIL8RA GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession B09989 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis JP08103276 of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity IL-8RA can be determinedusing assays known in the art: Matthews et al., in LymphokinesP25024/CXCR1_HUMAN and Interferens: A Practical Approach, Clemens etal., eds, IRL Press, Washington, C—X—C chemokine receptor D.C. 1987, pp.221-225; and Holmes et al (1991) Science 253, 1278-80. Soluble IL-8 type1 receptor A polypeptides may be useful for inhibiting interleukinactivities. (SEQ ID NO: 294) Human IL-8 receptor Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, proteinhIL8R GeneSeq monocytes, and macrophages. Known functions includestimulating proliferation of Accession B09990 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisJP08103276 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity IL-8RA can be determined using assaysknown in the art: Matthews et al., in Lymphokines P25024/CXCR1_HUMAN andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, C—X—C chemokine receptor D.C. 1987, pp. 221-225; and Holmeset al (1991) Science 253, 1278-80. Soluble IL-8 type 1 receptorpolypeptides may be useful for inhibiting interleukin activities. (SEQID NO: 294) IL-8RB P25025/CXCR2_HUMAN C—X—C chemokine receptor type 2(SEQ ID NO: 295) Interleukin-2 receptor Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, associatedprotein p43 monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis R97569WO9621732- of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity SEQ ID NO: 2 of can be determinedusing assays known in the art: Matthews et al., in Lymphokines WO9621732and Interferons: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (SEQ ID NO: 395) D.C. 1987, pp. 221-225; and Gillis et al(1978) J. Immunol. 120, 2027. Soluble IL-2 receptor polypeptides may beuseful for inhibiting interleukin activities. Human interleukin-17Interleukins are a group of multi-functional cytokines synthesized bylymphocytes, receptor GeneSeq monocytes, and macrophages. Knownfunctions include stimulating proliferation of Accession W04185 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis WO9629408 of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity Q96F46/I17RA_HUMAN can bedetermined using assays known in the art: Matthews et al., inLymphokines Interleukin-17 receptor A and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 290)D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86.Soluble IL- 17 receptor polypeptides may be useful for inhibitinginterleukin activities. Human interleukin-11 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, receptor GeneSeqmonocytes, and macrophages. Known functions include stimulatingproliferation of Accession R99090 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis WO9619574 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Q14626/I11RA_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Interleukin-11receptor and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, subunit alpha D.C. 1987, pp. 221-225; and Lu et al(1994) J immunol. Methods 173, 19. Soluble IL- (SEQ ID NO: 297) 11receptor polypeptides may be useful for inhibiting interleukinactivities. Human interleukin-1 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, receptoraccessory protein monocytes, and macrophages. Known functions includestimulating proliferation of GeneSeq Accession immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxis W01911WO9623067 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity Human IL1R Acp can be determined usingassays known in the art: Matthews et al., in Lymphokines SEQ ID NO: 3 ofand Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, WO9623067 D.C. 1987, pp. 221-225; and Orencole & Dinarello(1989) Cytokine 1, 14-20. (SEQ ID NO: 396) Inflammatory disordersimmunologic disorders, cancer Soluble Human IL1R Acp SEQ ID NO: 9 ofWO9623067 (SEQ ID NO: 397) AGF Protein GeneSeq Interleukins are a groupof multi-functional cytokines synthesized by lymphocytes, AccessionR92749 U.S. monocytes, and macrophages. Known functions includestimulating proliferation of Pat. No. 5,488,032 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisQ8NI99/ANGL6_HUMAN of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity Angiopoietin-related can bedetermined using assays known in the art: Matthews et al., inLymphokines protein 6 and Interferens: A Practical Approach, Clemens etal., eds, IRL Press, Washington, (SEQ ID NO: 278) D.C. 1987, pp.221-225. Inflammatory disorders immunologic disorders, cancer Humaninterleukin-1 type- Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, 3 receptor GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession R91064 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis W09607739 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activity SEQID NO: 2 and 4 of can be determined using assays known in the art:Matthews et al., in Lymphokines WO9607739 and Interferens: A PracticalApproach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 398and SEQ D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine1, 14-20. Soluble ID NO: 399, respectively) IL-type-3 receptorpolypeptides may be useful for inhibiting interleukin activities Humaninterleukin-13 beta Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, receptor GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession W24972 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis WO9720926 of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activity SEQID NO: 2 from can be determined using assays known in the art: Matthewset al., in Lymphokines WO9720926 and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 400) D.C. 1987,pp. 221-225; and Boutelier et al (1995) J. Immunol. Methods 181, 29.Soluble IL-13 beta receptor polypeptides may be useful for inhibitinginterleukin activities. Human interleukin-13 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, alpha receptorGeneSeq monocytes, and macrophages. Known functions include stimulatingproliferation of Accession W24973 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis WO9720926 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity IL-13RA1 can be determined using assays known inthe art: Matthews et al., in Lymphokines P78552/I13R1_HUMAN andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, Interleukin-13 receptor D.C. 1987, pp. 221-225; andBoutelier et al (1995) J. Immunol. Methods 181, 29. subunit alpha-1Soluble IL-13 alpha receptor polypeptides may be useful for inhibitinginterleukin (isoform 1) activities. (SEQ ID NO: 298) IL-13RA2Q14627/I13R2_HUMAN Interleukin-13 receptor subunit alpha-2 (SEQ ID NO:299) Human interleukin-4 Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, receptor GeneSeq monocytes, andmacrophages. Known functions include stimulating proliferation ofAccession W13499 U.S. immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis Pat. No. 5,599,905 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity P24394/IL4RA_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Interleukin-4 receptorand Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, subunit alpha D.C. 1987, pp. 221-225; and Siegel & Mostowski(1990) J Immunol Methods 132, (isoform 1) 287-295. Soluble IL-4 receptorpolypeptides may be useful for inhibiting interleukin (SEQ ID NO: 300)activities. Human interleukin-12 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, beta-2 receptorGeneSeq monocytes, and macrophages. Known functions include stimulatingproliferation of Accession W12771 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis EP759466 of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity Q9966/|I12R2_HUMAN can be determined using assays known in theart: Matthews et al., in Lymphokines Interleukin-12 receptor andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, subunit beta-2 D.C. 1987, pp. 221-225; and Hori et al(1987), Blood 70, 1069-1078. Soluble IL-12 (isoform 1) beta-2 receptorpolypeptides may be useful for inhibiting interleukin activities. (SEQID NO: 301) Human interleukin-12 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, beta-1 receptor.GeneSeq monocytes, and macrophages. Known functions include stimulatingproliferation of Accession W12772 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis EP759466 of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity P4270/|I12R1_HUMAN can be determined using assays known in theart: Matthews et al., in Lymphokines Interleukin-12 receptor andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, subunit beta-1 D.C. 1987, pp. 221-225; and Hori et al(1987), Blood 70, 1069-1078. Soluble IL-12 (isoform 1) beta-1 receptorpolypeptides may be useful for inhibiting interleukin activities. (SEQID NO: 302) Human IL-9 receptor Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, protein GeneSeqmonocytes, and macrophages. Known functions include stimulatingproliferation of Accessions W64055, immune cells (e.g., T helper cells,B cells, eosinophils, and lymphocytes), chemotaxis W64056, and W64057 ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity WO9824904 can be determined using assays known inthe art: Matthews et al., in Lymphokines Q01113/IL9R_HUMAN andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, Interleukin-9 receptor D.C. 1987, pp. 221-225; and Yang etal (1989) Blood 74, 1880-84. Soluble IL-9 (isoform 1) receptorpolypeptides may be useful for inhibiting interleukin activities. (SEQID NO: 303) IL-10 receptor GeneSeq Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, Accession W41804U.S. monocytes, and macrophages. Known functions include stimulatingproliferation of Pat. No. 5,716,804 immune cells (e.g., T helper cells,B cells, eosinophils, and lymphocytes), chemotaxis IL-10RA ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Q13651/I10R1_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Interleukin-10receptor and Interferens: A Practical Approach, Clemens et al., eds, IRLPress, Washington, subunit alpha D.C. 1987, pp. 221-225; andThompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. (SEQ ID NO: 304)Soluble IL-10 receptor polypeptides may be useful for inhibitinginterleukin IL-10RB activities. Q0833/|I10R2_HUMAN Interleukin-10receptor subunit beta (SEQ ID NO: 305) Human IL-6 receptor Interleukinsare a group of multi-functional cytokines synthesized by lymphocytes,GeneSeq Accession monocytes, and macrophages. Known functions includestimulating proliferation of Y30938 JP11196867 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisP08887/IL6RA_HUMAN of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity Interleukin-6 receptor can bedetermined using assays known in the art: Matthews et al., inLymphokines subunit alpha and Interferens: A Practical Approach, Clemenset al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225;and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. (SEQ ID NO: 306)Soluble IL-6 receptor polypeptides may be useful for inhibitinginterleukin activities. Il-17 receptor GeneSeq Interleukins are a groupof multi-functional cytokines synthesized by lymphocytes, AccessionY97181 U.S. monocytes, and macrophages. Known functions includestimulating proliferation of Pat. No. 6,096,305 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisQ96F46/I17RA_HUMAN of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity Interleukin-17 receptor A can bedetermined using assays known in the art: Matthews et al., inLymphokines (SEQ ID NO: 290) and Interferens: A Practical Approach,Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; andYao et al (1995) J. Immunol. 155, 5483-86. Soluble IL- 17 receptorpolypeptides may be useful for inhibiting interleukin activities. Il-17receptor GeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession Y97131 U.S. monocytes, andmacrophages. Known functions include stimulating proliferation of Pat.No. 6,100,235 immune cells (e.g., T helper cells, B cells, eosinophils,and lymphocytes), chemotaxis Q96F46/I17RA_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-17 receptor A can be determined using assays known in theart: Matthews et al., in Lymphokines (SEQ ID NO: 290) and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington, D.C.1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86.Soluble IL- 17 receptor polypeptides may be useful for inhibitinginterleukin activities. Human interleukin-3 Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, receptor GeneSeqmonocytes, and macrophages. Known functions include stimulatingproliferation of Accession R25300 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis EP509826 of neutrophilsand T lymphocytes, and/or inhibition of interferons. Interleukinactivity P26951/IL3RA_HUMAN can be determined using assays known in theart: Matthews et al., in Lymphokines Interleukin-3 receptor andInterferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, subunit alpha D.C. 1987, pp. 221-225; Kitamura et al (1989)J Cell Physiol. 140 323-334. Soluble (isoform 1) IL-3 receptorpolypeptides may be useful for inhibiting interleukin activities. (SEQID NO: 307) Human GM-CSF receptor Interleukins are a group ofmulti-functional cytokines synthesized by lymphocytes, GeneSeq Accessionmonocytes, and macrophages. Known functions include stimulatingproliferation of R10919 WO9102063 immune cells (e.g., T helper cells, Bcells, eosinophils, and lymphocytes), chemotaxis GM-CSF receptor A ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity P15509/CSF2R_HUMAN can be determined using assaysknown in the art: Matthews et al., in Lymphokines Granulocyte-macrophageand Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, colony-stimulating factor D.C. 1987, pp. 221-225. SolubleGM-CSF receptor polypeptides may be useful for receptor subunit alphainhibiting interleukin activities. (isoform 1) (SEQ ID NO: 308) GM-CSFreceptor B P32927/IL3RB_HUMAN Cytokine receptor common subunit beta(isoform 1) (SEQ ID NO: 309) Human IL-5 receptor alpha Interleukins area group of multi-functional cytokines synthesized by lymphocytes, chainGeneSeq Accession monocytes, and macrophages. Known functions includestimulating proliferation of R25064 EP492214 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisQ01344/IL5RA_HUMAN of neutrophils and T lymphocytes, and/or inhibitionof interferons. Interleukin activity Interleukin-5 receptor can bedetermined using assays known in the art: Matthews et al., inLymphokines subunit alpha and Interferens: A Practical Approach, Clemenset al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225;Kitamura et al (1989) J Cell Physiol. 140 323-334. Soluble (SEQ ID NO:310) IL-5 receptor alpha polypeptides may be useful for inhibitinginterleukin activities. Il-5 receptor GeneSeq Interleukins are a groupof multi-functional cytokines synthesized by lymphocytes, AccessionW82842 monocytes, and macrophages. Known functions include stimulatingproliferation of WO9847923 immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis Q01344/IL5RA_HUMAN ofneutrophils and T lymphocytes, and/or inhibition of interferons.Interleukin activity Interleukin-5 receptor can be determined usingassays known in the art: Matthews et al., in Lymphokines subunit alphaand Interferens: A Practical Approach, Clemens et al., eds, IRL Press,Washington, (isoform 1) D.C. 1987, pp. 221-225; Kitamura et al (1989) JCell Physiol. 140 323-334. Soluble (SEQ ID NO: 310) IL-5 receptorpolypeptides may be useful for inhibiting interleukin activities. Il-6receptor GeneSeq Interleukins are a group of multi-functional cytokinessynthesized by lymphocytes, Accession R37215 monocytes, and macrophages.Known functions include stimulating proliferation of JP05091892 immunecells (e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis P08887/IL6RA_HUMAN of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity Interleukin-6 receptorcan be determined using assays known in the art: Matthews et al., inLymphokines subunit alpha and Interferens: A Practical Approach, Clemenset al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225;and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. (SEQ ID NO: 306)Soluble IL-6 receptor polypeptides may be useful for inhibitinginterleukin activities. Human B cell stimulating Interleukins are agroup of multi-functional cytokines synthesized by lymphocytes, factor-2receptor GeneSeq monocytes, and macrophages. Known functions includestimulating proliferation of Accession P90525 immune cells (e.g., Thelper cells, B cells, eosinophils, and lymphocytes), chemotaxisAU8928720 of neutrophils and T lymphocytes, and/or inhibition ofinterferons. Interleukin activity P08887/IL6RA_HUMAN can be determinedusing assays known in the art: Matthews et al., in LymphokinesInterleukin-6 receptor and Interferens: A Practical Approach, Clemens etal., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225.Soluble B cell stimulating factor-2 receptor polypeptides (isoform 1)may be useful for inhibiting interleukin activities. (SEQ ID NO: 306)IL-7 receptor clone Interleukins are a group of multi-functionalcytokines synthesized by lymphocytes, GeneSeq Accession monocytes, andmacrophages. Known functions include stimulating proliferation of R08330EP403114 immune cells (e.g., T helper cells, B cells, eosinophils, andlymphocytes), chemotaxis P1687/|IL7RA_HUMAN of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activityInterleukin-7 receptor can be determined using assays known in the art:Matthews et al., in Lymphokines subunit alpha and Interferens: APractical Approach, Clemens et al., eds, IRL Press, Washington,(isoform 1) D.C. 1987, pp. 221-225; and Park et al (1990) J. Exp. Med.171, 1073-79. Soluble IL- (SEQ ID NO: 311) 7 receptor polypeptides maybe useful for inhibiting interleukin activities. EPO receptor; EPOR EPOReceptor is involved in the proliferation and differentiation oferythroblasts. EPO GeneSeq Accession Receptor activity can be determinedusing assays known in the art, such as, J Biol R06512 WO9008822 Chem2001 Mar. 23; 276(12: 8995-9002; JAK2 protein tyrosine kinase activity:Blood P19235/EPOR_HUMAN 1994 Sep. 1; 84(5): 1501-7 and Mol Cell Biol.1994 Oct; 14(10: 6506-14. Inflammatory Erythropoietin receptordisorders, immunologic disorders, cancer, erythroblast proliferation anddifferentiation (isoform EPOR-F) (SEQ ID NO: 312) IL-15 receptor GeneSeqInterleukins are a group of multi-functional cytokines synthesized bylymphocytes, Accession R90843 monocytes, and macrophages. Knownfunctions include stimulating proliferation of WO9530695 immune cells(e.g., T helper cells, B cells, eosinophils, and lymphocytes),chemotaxis Q1326/|I15RA_HUMAN of neutrophils and T lymphocytes, and/orinhibition of interferons. Interleukin activity Interleukin-15 receptorcan be determined using assays known in the art: Matthews et al., inLymphokines subunit alpha and Interferens: A Practical Approach, Clemenset al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225;and Giri et al (1994) EMBO J. 13 2822-2830. Soluble IL-15 (SEQ ID NO:313) receptor polypeptides may be useful for inhibiting interleukinactivities. Obesity. Metabolic Disease. Diabetes. Enhancing secretionand stability of Interleukin 15 CD137; 4-1BB Receptor Activitiesassociated with apoptosis, NF-kB activation, and co-stimulation ofimmune Protein GeneSeq cells such as T and B cells. Apoptosis activity,NF-kB activation, and B and T cell co- Accession R70977 stimulation canbe determined using assays known in the art: Moore et al., 1999,WO9507984 Science, 285(5425): 260-3; Song HY et al., 1997 Proc Natl AcadSci USA 94(18): Q07011/TNR9_HUMAN 9792-6; Epsevik and Nissen-Meyer,1986, J. Immunol. Methods. Soluble 4-1BB Tumor necrosis factor receptorpolypeptides may be useful for inhibiting apoptosis, NF-kB activation,receptor superfamily and/or co-stimulation of immune cells such as B andT cells. member 9 (SEQ ID NO: 314) BCMA GeneSeq Activities associatedwith apoptosis, NF-kB activation, and co-stimulation of immune AccessionY71979 cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- WO0068378 stimulation can be determinedusing assays known in the art: Moore et al., 1999, Q02223/TNR17_HUMANScience, 285(5425): 260-3; Song HY et al., 1997 Proc Natl Acad Sci USA94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J.Immunol. Methods. Soluble BCMA receptor superfamily receptorpolypeptides may be useful for inhibiting apoptosis, NF-kB activation,member 17 and/or co-stimulation of immune cells such as B and T cells.(isoform 1) (SEQ ID NO: 315) CD27 GeneSeq Accession Activitiesassociated with apoptosis, NF-kB activation, and co-stimulation ofimmune R20814 WO9201049 cells such as T and B cells. Apoptosis activity,NF-kB activation, and B and T cell co- P26842/CD27_HUMAN stimulation canbe determined using assays known in the art: Moore et al., 1999, CD27antigen Science, 285(5425): 260-3; Song HY et al., 1997 Proc Natl AcadSci USA 94(18): (SEQ ID NO: 316) 9792-6; Epsevik and Nissen-Meyer, 1986,J. Immunol. Methods. Soluble CD27 polypeptides may be useful forinhibiting apoptosis, NF-kB activation, and/or co- stimulation of immunecells such as B and T cells. CD30 GeneSeq Accession Activitiesassociated with apoptosis, NF-kB activation, and co-stimulation ofimmune R35478 DE4200043 cells such as T and B cells. Apoptosis activity,NF-kB activation, and B and T cell co- P28908/TNR8_HUMAN stimulation canbe determined using assays known in the art: Moore et al., 1999, Tumornecrosis factor Science, 285(5425): 260-3; Song HY et al., 1997 ProcNatl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik andNissen-Meyer, 1986, J. Immunol. Methods. Soluble CD30 member 8polypeptides may be useful for inhibiting apoptosis, NF-kB activation,and/or co- (isoform 1) stimulation of immune cells such as B and Tcells. (SEQ ID NO: 317) CD40 GeneSeq Accession Activities associatedwith apoptosis, NF-kB activation, and co-stimulation of immune Y33499WO9945944 cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- P25942/TNR5_HUMAN stimulation can bedetermined using assays known in the art: Moore et al., 1999, Tumornecrosis factor Science, 285(5425): 260-3; Song HY et al., 1997 ProcNatl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik andNissen-Meyer, 1986, J. Immunol. Methods. Soluble CD40 member 5polypeptides may be useful for inhibiting apoptosis, NF-kB activation,and/or co- (isoform 1) stimulation of immune cells such as B and Tcells. (SEQ ID NO: 318) EDAR Genbank Activities associated withapoptosis, NF-kB activation, and co-stimulation of immune AccessionAAD50077 cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- Q9UNE0|EDAR_HUMAN stimulation can bedetermined using assays known in the art: Moore et al., 1999, Tumornecrosis factor Science, 285(5425): 260-3; Song HY et al., 1997 ProcNatl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik andNissen-Meyer, 1986, J. Immunol. Methods. Immune Disorders, member EDARLymphomas, X-linked hypohidrotic ectodermal dysplasia (isoform 1) (SEQID NO: 319) OX40; ACT-4 GeneSeq Activities associated with apoptosis,NF-kB activation, and co-stimulation of immune Accession R74737 cellssuch as T and B cells. Apoptosis activity, NF-kB activation, and B and Tcell co- WO9512673 stimulation can be determined using assays known inthe art: Moore et al., 1999, P43489/TNR4_HUMAN Science, 285(5425):260-3; Song HY et al., 1997 Proc Natl Acad Sci USA 94(18): Tumornecrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol.Methods. Immune Disorders, receptor superfamily Lymphomas, T celldisorders member 4 (SEQ ID NO: 320) TACI GeneSeq Accession Activitiesassociated with apoptosis, NF-kB activation, and co-stimulation ofimmune W75783 WO9839361 cells such as T and B cells. Apoptosis activity,NF-kB activation, and B and T cell co- O14836/TR13B_HUMAN stimulationcan be determined using assays known in the art: Moore et al., 1999,Tumor necrosis factor Science, 285(5425): 260-3; Song HY et al., 1997Proc Natl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik andNissen-Meyer, 1986, J. Immunol. Methods. Soluble TACI member 13Breceptor polypeptides may be useful for inhibiting apoptosis, NF-kBactivation, (isoform 1) and/or co-stimulation of immune cells such as Band T cells. (SEQ ID NO: 321) TNF-R GeneSeq Activities associated withapoptosis, NF-kB activation, and co-stimulation of immune AccessionR10986 cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- AU9058976 stimulation can be determinedusing assays known in the art: Moore et al., 1999, P19438/TNR1A_HUMANScience, 285(5425): 260-3; Song HY et al., 1997 Proc Natl Acad Sci USA94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J.Immunol. Methods. Soluble TNF-R receptor superfamily receptorpolypeptides may be useful for inhibiting apoptosis, NF-kB activation,member 1A and/or co-stimulation of immune cells such as B and T cells.(isoform 1) (SEQ ID NO: 322) TNF-RII; TNF p75 Activities associated withapoptosis, NF-kB activation, and co-stimulation of immune receptor;Death Receptor cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- GeneSeq Accession stimulation can bedetermined using assays known in the art: Moore et al., 1999, R11141EP418014 Science, 285(5425): 260-3; Song HY et al., 1997 Proc Natl AcadSci USA 94(18): P20333/TNR1B_HUMAN 9792-6; Epsevik and Nissen-Meyer,1986, J. Immunol. Methods. Soluble TNFR-II Tumor necrosis factorreceptor polypeptides may be useful for inhibiting apoptosis, NF-kBactivation, receptor superfamily and/or co-stimulation of immune cellssuch as B and T cells. member 1B (isoform 1) (SEQ ID NO: 323) hAPO-4;TROY GeneSeq Activities associated with apoptosis, NF-kB activation, andco-stimulation of immune Accession W93581 cells such as T and B cells.Apoptosis activity, NF-kB activation, and B and T cell co- WO9911791stimulation can be determined using assays known in the art: Moore etal., 1999, Q9N568/TNR19_HUMAN Science, 285(5425): 260-3; Song HY et al.,1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6;Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Immune Disorders,receptor superfamily Cancers member 19 (isoform 1) (SEQ ID NO: 324)TNF-alpha precursor Activities associated with apoptosis, NF-kBactivation, and co-stimulation of immune GeneSeq Accession cells such asT and B cells. Apoptosis activity, NF-kB activation, and B and T cellco- P60074 EP205038 stimulation can be determined using assays known inthe art: Moore et al., 1999, Science, 285(5425): 260-3; Song HY et al.,1997 Proc Natl Acad Sci USA 94(18): 9792-6; Epsevik and Nissen-Meyer,1986, J. Immunol. Methods. Inflammatory disorders immunologic disorders,cancer Human TNF-alpha Activities associated with apoptosis, NF-kBactivation, and co-stimulation of immune GeneSeq Accession cells such asT and B cells. Apoptosis activity, NF-kB activation, and B and T cellco- R62463 EP619372 stimulation can be determined using assays known inthe art: Moore et al., 1999, P01375/TNFA_HUMAN Science, 285(5425):260-3; Song HY et al., 1997 Proc Natl Acad Sci USA 94(18): Tumornecrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol.Methods. Inflammatory (SEQ ID NO: 325) disorders immunologic disorders,cancer Human TNF-alpha Activities associated with apoptosis, NF-kBactivation, and co-stimulation of immune GeneSeq Accession cells such asT and B cells. Apoptosis activity, NF-kB activation, and B and T cellco- R42679 EP563714 stimulation can be determined using assays known inthe art: Moore et al., 1999, P01375/TNFA_HUMAN Science, 285(5425):260-3; Song HY et al., 1997 Proc Natl Acad Sci USA 94(18): Tumornecrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol.Methods. Inflammatory (SEQ ID NO: 325) disorders immunologic disorders,cancer Human TNF-beta (LT- Activities associated with apoptosis, NF-kBactivation, and co-stimulation of immune alpha) GeneSeq cells such as Tand B cells. Apoptosis activity, NF-kB activation, and B and T cell co-Accession B37799 stimulation can be determined using assays known in theart: Moore et al., 1999, WO0064479 Science, 285(5425): 260-3; Song HY etal., 1997 Proc Natl Acad Sci USA 94(18): P01374/TNFB_HUMAN 9792-6;Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. InflammatoryLymphotoxin-alpha disorders immunologic disorders, cancer (SEQ ID NO:326) LT-alpha GeneSeq Activities associated with apoptosis, NF-kBactivation, and co-stimulation of immune Accession P70107 cells such asT and B cells. Apoptosis activity, NF-kB activation, and B and T cellco- EP250000 stimulation can be determined using assays known in theart: Moore et al., 1999, P01374/TNFB_HUMAN Science, 285(5425): 260-3;Song HY et al., 1997 Proc Natl Acad Sci USA 94(18): Lymphotoxin-alpha9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods.Inflammatory (SEQ ID NO: 326) disorders immunologic disorders, cancerLT-beta GeneSeq Activities associated with apoptosis, NF-kB activation,and co-stimulation of immune Accession R56869 cells such as T and Bcells. Apoptosis activity, NF-kB activation, and B and T cell co-WO9413808 stimulation can be determined using assays known in the art:Moore et al., 1999, Q06643/TNFC_HUMAN Science, 285(5425): 260-3; Song HYet al., 1997 Proc Natl Acad Sci USA 94(18): Lymphotoxin-beta 9792-6;Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory(isoform 1) disorders immunologic disorders, cancer (SEQ ID NO: 327)OPGL GeneSeq Activities associated with apoptosis, NF-kB activation, andco-stimulation of immune Accession W83195 cells such as T and B cells.Apoptosis activity, NF-kB activation, and B and T cell co- WO9846751stimulation can be determined using assays known in the art: Moore etal., 1999, O14788/TNF11_HUMAN Science, 285(5425): 260-3; Song HY et al.,1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6;Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory ligandsuperfamily disorders immunologic disorders, cancer, loss of bone massmember 11 (isoform 1) (SEQ ID NO: 328) FasL GeneSeq Accession Activitiesassociated with apoptosis, NF-kB activation, and co-stimulation ofimmune W98071 WO9903999 cells such as T and B cells. Apoptosis activity,NF-kB activation, and B and T cell co- P48023/TNFL6_HUMAN stimulationcan be determined using assays known in the art: Moore et al., 1999,Tumor necrosis factor Science, 285(5425): 260-3; Song HY et al., 1997Proc Natl Acad Sci USA 94(18): ligand superfamily 9792-6; Epsevik andNissen-Meyer, 1986, J. Immunol. Methods. Inflammatory member 6 disordersimmunologic disorders, cancer (isoform 1) (SEQ ID NO: 329) FasL GeneSeqAccession Activities associated with apoptosis, NF-kB activation, andco-stimulation of immune W95041 WO9903998 cells such as T and B cells.Apoptosis activity, NF-kB activation, and B and T cell co-P48023/TNFL6_HUMAN stimulation can be determined using assays known inthe art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425):260-3; Song HY et al., 1997 Proc Natl Acad Sci USA 94(18): ligandsuperfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods.Inflammatory member 6 disorders immunologic disorders, cancer(isoform 1) (SEQ ID NO: 329) CD27L GeneSeq Activities associated withapoptosis, NF-kB activation, and co-stimulation of immune AccessionR50121 cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- WO9405691 stimulation can be determinedusing assays known in the art: Moore et al., 1999, P32970/CD70_HUMANScience, 285(5425): 260-3; Song HY et al., 1997 Proc Natl Acad Sci USA94(18): CD70 antigen 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol.Methods. Inflammatory (isoform 1) disorders immunologic disorders,cancer (SEQ ID NO: 330) CD30 ligand GeneSeq Activities associated withapoptosis, NF-kB activation, and co-stimulation of immune AccessionR45007 cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- WO9324135 stimulation can be determinedusing assays known in the art: Moore et al., 1999, P32971/TNFL8_HUMANScience, 285(5425): 260-3; Song HY et al., 1997 Proc Natl Acad Sci USA94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J.Immunol. Methods. Inflammatory ligand superfamily disorders immunologicdisorders, cancer member 8 (SEQ ID NO: 331) CD40L GeneSeq Activitiesassociated with apoptosis, NF-kB activation, and co-stimulation ofimmune Accession R85486 cells such as T and B cells. Apoptosis activity,NF-kB activation, and B and T cell co- WO9529935 stimulation can bedetermined using assays known in the art: Moore et al., 1999,P29965/CD40L_HUMAN Science, 285(5425): 260-3; Song HY et al., 1997 ProcNatl Acad Sci USA 94(18): CD40 ligand 9792-6; Epsevik and Nissen-Meyer,1986, J. Immunol. Methods. Inflammatory (SEQ ID NO: 332) disordersimmunologic disorders, cancer 4-1BB ligand GeneSeq Activities associatedwith apoptosis, NF-kB activation, and co-stimulation of immune AccessionW26657 U.S. cells such as T and B cells. Apoptosis activity, NF-kBactivation, and B and T cell co- Pat. No. 5,674,704 stimulation can bedetermined using assays known in the art: Moore et al., 1999,P41273/TNFL9_HUMAN Science, 285(5425): 260-3; Song HY et al., 1997 ProcNatl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik andNissen-Meyer, 1986, J. Immunol. Methods. Inflammatory ligand superfamilydisorders immunologic disorders, cancer member 9 (SEQ ID NO: 333) FASLigand Inhibitory Activities associated with apoptosis, NF-kBactivation, and co-stimulation of immune Protein (DcR3) GeneSeq cellssuch as T and B cells. Apoptosis activity, NF-kB activation, and B and Tcell co- Accession B19335 stimulation can be determined using assaysknown in the art: Moore et al., 1999, WO0058465 Science, 285(5425):260-3; Song HY et al., 1997 Proc Natl Acad Sci USA 94(18):O95407/TNF6B_HUMAN 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol.Methods. Soluble DcR3 Tumor necrosis factor polypeptides may be usefulfor inhibiting apoptosis, NF-kB activation, and/or co- receptorsuperfamily stimulation of immune cells such as B and T cells. member 6B(SEQ ID NO: 334) OX40L GeneSeq Activities associated with apoptosis,NF-kB activation, and co-stimulation of immune Accession R79903 cellssuch as T and B cells. Apoptosis activity, NF-kB activation, and B and Tcell co- WO9521915 stimulation can be determined using assays known inthe art: Moore et al., 1999, P23510/TNFL4_HUMAN Science, 285(5425):260-3; Song HY et al., 1997 Proc Natl Acad Sci USA 94(18): Tumornecrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol.Methods. Inflammatory ligand superfamily disorders immunologicdisorders, cancer member 4 (isoform 1) (SEQ ID NO: 335) Proteaseinhibitor peptides Peptides that inhibit the function/binding of HIV HIVprotease activities are known in GeneSeq Accessions the art: HIVprotease assays: EP0387231. One can modify the assay to look for R12435,R12436, R12437, inhibition using any of the disclosed protease inhibitorpolypeptides. HIV, R12438, R12439, R12440, inflammatory disorders,immunologic disorders, cancer, viral infections and R1244 WO9106561Retroviral protease Peptides that inhibit the function/binding of HIVHIV protease activities are known in inhibitors GeneSeq the art: HIVprotease assays: EP0387231. One can modify the assay to look forAccessions R06660, inhibition using any of the disclosed proteaseinhibitor polypeptides. HIV, R06661, R06662, R06663, inflammatorydisorders, immunologic disorders, cancer, viral infections R06664,R06665, R06666, R06667, R06668, R06669, R06670, R06671, R06672, R06673,R06674, R06675, and R06676 EP387231 HIV protease inhibiting Peptidesthat inhibit the function/binding of HIV HIV protease activities areknown in peptides GeneSeq the art: HIV protease assays: EP0387231. Onecan modify the assay to look for Accessions R59293, inhibition using anyof the disclosed protease inhibitor polypeptides. HIV, R59294, R59295,R59296, inflammatory disorders, immunologic disorders, cancer, viralinfections R59297, R59298, R59299, R592300, R59301, R59302, R59301,R59302, R59303, R59304, R59305, R59306, R59307, R59308, R59309, R59310,R59311, R59312, R59313, R59314, R59315, R59316, R59317 R59318, R59319,R59320, R59321, R59322, R59323, R59324, R59325, R59326, R59327, R59328,R59329, R59330, R59331, R59332, R59333, R59334, R59335, R59336, R59337,R59338, R59339, R59340, R59341, R59342, R59343, R59344, R59345, R59346,R59347, R59348, R59349, and R59350 WO9301828 HIV-1 protease inhibitorsPeptides that inhibit the function/binding of HIV HIV proteaseactivities are known in GeneSeq Accessions the art: HIV protease assays:EP0387231. One can modify the assay to look for R86326, R86327, R86328,inhibition using any of the disclosed protease inhibitor polypeptides.HIV, R86329, R86330, R86331, inflammatory disorders, immunologicdisorders, cancer, viral infections R86332, R86333, R86334, R86335,R86336, R86337, R86338, R86339, R86340, R86341, R86342, R86343, R86344,R86345, R86346, R86347, R86348, R86349, R86350, R86351, R86352, R86353,R86354, R86355, R86356, R86357, R86358, R86359, R86360, R86361, R86362,R86363, R86364, R86365, R86366, R86367, R86368, R86369, R86370, andR86371 DE4412174 HIV Inhibitor Peptide Peptides that inhibit thefunction/binding of HIV HIV protease activities are known in GeneSeqAccession the art: HIV protease assays: EP0387231. One can modify theassay to look for Y89687 WO9959615 inhibition using any of the disclosedprotease inhibitor polypeptides. HIV, inflammatory disorders,immunologic disorders, cancer, viral infections HIV Inhibitor PeptidePeptides that inhibit the function/binding of HIV HIV proteaseactivities are known in GenSeq Accession the art: HIV protease assays:EP0387231. One can modify the assay to look for Y31955 WO9948513inhibition using any of the disclosed protease inhibitor polypeptides.HIV, inflammatory disorders, immunologic disorders, cancer, viralinfections HIV Inhibitor Peptide Peptides that inhibit thefunction/binding of HIV HIV protease activities are known in Science291, 884 (2001); the art: HIV protease assays: EP0387231. One can modifythe assay to look for Published online 12 Jan. inhibition using any ofthe disclosed protease inhibitor polypeptides. HIV, 2001;10.1126/science.1057453 inflammatory disorders, immunologic disorders,cancer, viral infections Human monocyte Chemokines are a family ofrelated small, secreted proteins involved in biological chemoattractantfactor processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. hMCP-3 GeneSeq Members of this family are involved in asimilarly diverse range of pathologies Accession R73915 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The WO9509232 chemokines exert their effects by acting on afamily of seven transmembrane G- P80098/CCL7_HUMAN C- protein coupledreceptors. Over 40 human chemokines have been described, which C motifchemokine 7 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 336) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders, particularly useful fortreating bacterial and/or viral menigitis Human monocyte Chemokines area family of related small, secreted proteins involved in biologicalchemoattractant factor processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. hMCP-1 GeneSeq Members of thisfamily are involved in a similarly diverse range of pathologiesAccession R73914 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The WO9509232 chemokines exert theireffects by acting on a family of seven transmembrane G-P13500/CCL2_HUMAN C- protein coupled receptors. Over 40 human chemokineshave been described, which C motif chemokine 2 bind to ~17 receptorsthus far identified. Chemokine activities can be determined (SEQ ID NO:337) using assays known in the art: Methods in Molecular Biology, 2000,vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders,particularly useful for treating bacterial and/or viral menigitis Humangro-beta Chemokines are a family of related small, secreted proteinsinvolved in biological chemokine GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. AccessionsR66699 and Members of this family are involved in a similarly diverserange of pathologies W17671 WO9429341 including inflammation, allergy,tissue rejection, viral infection, and tumor biology. TheP19875/CXCL2_HUMAN chemokines exert their effects by acting on a familyof seven transmembrane G- C—X—C motif chemokine 2 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 338) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune disorders, inflammatory disorders, blood- related disorders,stem cell transplantation, cancer Human gro-gamma Chemokines are afamily of related small, secreted proteins involved in biologicalchemokine GeneSeq processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. Accessions R66700 and Members of this familyare involved in a similarly diverse range of pathologies W17672WO9429341 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P19876/CXCL3_HUMAN chemokines exerttheir effects by acting on a family of seven transmembrane G- C—X—Cmotif chemokine 3 protein coupled receptors. Over 40 human chemokineshave been described, which (SEQ ID NO: 339) bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders,inflammatory disorders, blood- related disorders, stem celltransplantation, cancer Human gro-alpha Chemokines are a family ofrelated small, secreted proteins involved in biological chemokineGeneSeq processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Accessions R66698 and Members of this family areinvolved in a similarly diverse range of pathologies W18024 WO9429341including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The P09341/GROA_HUMAN chemokines exert their effects byacting on a family of seven transmembrane G- Growth-regulated alphaprotein coupled receptors. Over 40 human chemokines have been described,which protein bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 340) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders, inflammatory disorders, blood-related disorders, stem cell transplantation, cancer Human eosinophil-Chemokines are a family of related small, secreted proteins involved inbiological expressed chemokine processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. (EEC) GeneSeq Members of thisfamily are involved in a similarly diverse range of pathologiesAccession WO5186 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The WO9632481 chemokines exert theireffects by acting on a family of seven transmembrane G- SEQ ID NO: 2 ofprotein coupled receptors. Over 40 human chemokines have been described,which WO9632481 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 401) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders, particularly treatment ofeosinophilia, inflammation, allergies, asthma, leukaemia and lymphomaChemokine-like protein Chemokines are a family of related small,secreted proteins involved in biological PF4-414 Full-Length andprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. Mature GeneSeq Members of this family are involved in asimilarly diverse range of pathologies Accessions R92318 and includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The R99809 WO9613587 chemokines exert their effects by actingon a family of seven transmembrane G- FIG. 3C of WO9613587 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 402) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Cancer and blood-related disorders, particularlymyelosuppression Chemokine-like protein IL- Chemokines are a family ofrelated small, secreted proteins involved in biological 8M3 GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. R99812 WO9613587 Members of this family areinvolved in a similarly diverse range of pathologies includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The chemokines exert their effects by acting on a family ofseven transmembrane G- protein coupled receptors. Over 40 humanchemokines have been described, which bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science253, 1278-80. Cancer and blood-related disorders, particularlymyelosuppression Human interleukin-8 (IL-8) Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. R99814 WO9613587 Members of this family areinvolved in a similarly diverse range of pathologies P10145/IL8_HUMANincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The Interleukin-8 chemokines exert their effects byacting on a family of seven transmembrane G- (isoform 1) protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 341) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer andblood-related disorders, particularly myelosuppression Chemokine-likeprotein IL- Chemokines are a family of related small, secreted proteinsinvolved in biological 8M1 Full-Length and processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeqMembers of this family are involved in a similarly diverse range ofpathologies Accessions R99815 and including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The R99803WO9613587 chemokines exert their effects by acting on a family of seventransmembrane G- FIG. 4B of WO9613587 protein coupled receptors. Over 40human chemokines have been described, which (SEQ ID NO: 403) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991)Science 253, 1278-80. Cancer and blood-related disorders, particularlymyelosuppression Chemokine-like protein IL- Chemokines are a family ofrelated small, secreted proteins involved in biological 8M8 Full-Lengthand processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. Mature GeneSeq Members of this family are involved in asimilarly diverse range of pathologies Accessions R99816 and includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The R99805 WO9613587 chemokines exert their effects by actingon a family of seven transmembrane G- FIG. 4C of WO9613587 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer andblood-related disorders, particularly myelosuppression Chemokine-likeprotein IL- Chemokines are a family of related small, secreted proteinsinvolved in biological 8M8 Full-Length and processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeqMembers of this family are involved in a similarly diverse range ofpathologies Accessions R99817 and including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The R99806WO9613587 chemokines exert their effects by acting on a family of seventransmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40human chemokines have been described, which (SEQ ID NO: 404) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991)Science 253, 1278-80. Cancer and blood-related disorders, particularlymyelosuppression. Chemokine-like protein IL- Chemokines are a family ofrelated small, secreted proteins involved in biological 8M8 Full-Lengthand processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. Mature GeneSeq Members of this family are involved in asimilarly diverse range of pathologies Accessions R99818 and includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The R99804 WO9613587 chemokines exert their effects by actingon a family of seven transmembrane G- FIG. 4C of WO9613587 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer andblood-related disorders, particularly myelosuppression. Chemokine-likeprotein IL- Chemokines are a family of related small, secreted proteinsinvolved in biological 8M8 Full-Length and processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeqMembers of this family are involved in a similarly diverse range ofpathologies Accessions R99819 and including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The R99807WO9613587 chemokines exert their effects by acting on a family of seventransmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40human chemokines have been described, which (SEQ ID NO: 404) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and blood-relateddisorders, particularly myelosuppression. Chemokine-like protein IL-Chemokines are a family of related small, secreted proteins involved inbiological 8M8 Full-Length and processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of thisfamily are involved in a similarly diverse range of pathologiesAccessions R99822 and including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The R9807 WO9613587 chemokines exerttheir effects by acting on a family of seven transmembrane G- FIG. 4C ofWO9613587 protein coupled receptors. Over 40 human chemokines have beendescribed, which (SEQ ID NO: 404) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Cancer and blood-relateddisorders, particularly myelosuppression. Human foetal spleen expressedChemokines are a family of related small, secreted proteins involved inbiological chemokine, processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. FSEC GeneSeq Members of thisfamily are involved in a similarly diverse range of pathologiesAccession R98499 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The WO9622374 chemokines exert theireffects by acting on a family of seven transmembrane G- SEQ ID NO: 2 ofprotein coupled receptors. Over 40 human chemokines have been described,which WO9622374 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 405) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders Liver expressed Chemokines area family of related small, secreted proteins involved in biologicalchemokine-1(LVEC-1) processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies R95689 WO9616979including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 2 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9616979 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 406) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inflammation of the liver Liver expressed Chemokines are a family ofrelated small, secreted proteins involved in biologicalchemokine-2(LVEC-2) processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies R95690 WO9616979including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 4 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9616979 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 407) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inflammation of the liver Pituitary expressed Chemokines are afamily of related small, secreted proteins involved in biologicalchemokine (PGEC) processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies R95691 WO9616979including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 6 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9616979 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 408) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inflammation, particularly of the liver Adenoid-expressed Chemokinesare a family of related small, secreted proteins involved in biologicalchemokine (ADEC) processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies R97664 WO9617868including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 2 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9617868 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 409) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inflammation, angiogenesis, tumorigenesis, musculoskeletal disordersHuman chemokine CC-2 Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W38170 WO9741230Members of this family are involved in a similarly diverse range ofpathologies Q16663/CCL15_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C-C motif chemokine15 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 342) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cellmigration, proliferation, and differentiation disorders Human chemokineHCC-1 Chemokines are a family of related small, secreted proteinsinvolved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W38171 WO9741230Members of this family are involved in a similarly diverse range ofpathologies Q16627/CCL14_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C-C motif chemokine14 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 343) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cellmigration, proliferation, and differentiation disorders Human chemokineCC-3 Chemokines are a family of related small, secreted proteinsinvolved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W38172 WO9741230Members of this family are involved in a similarly diverse range ofpathologies Q16627/CCL14_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C-C motif chemokine14 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 343) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cellmigration, proliferation, and differentiation Novel betachemokineChemokines are a family of related small, secreted proteins involved inbiological designated PTEC processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. GeneSeq Accession Members ofthis family are involved in a similarly diverse range of pathologiesW27271 WO9739126 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exerttheir effects by acting on a family of seven transmembrane G- WO9739126protein coupled receptors. Over 40 human chemokines have been described,which (SEQ ID NO: 410) bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders, vascular disorders, cancerHuman CX3C 111 amino Chemokines are a family of related small, secretedproteins involved in biological acid chemokine GeneSeq processes rangingfrom hematopoiesis, angiogenesis, and leukocyte trafficking. AccessionW23344 Members of this family are involved in a similarly diverse rangeof pathologies WO9727299 including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The SEQ ID NO: 2 ofchemokines exert their effects by acting on a family of seventransmembrane G- WO9727299 protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 411) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders,inflammatory diseases, abnormal proliferation, regeneration,degeneration, and atrophy Human CCF18 chemokine Chemokines are a familyof related small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. W25942 WO9721812 Members of this family areinvolved in a similarly diverse range of pathologies SEQ ID NO: 4 ofincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The WO9721812 chemokines exert their effects by acting ona family of seven transmembrane G- (SEQ ID NO: 412) protein coupledreceptors. Over 40 human chemokines have been described, which bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Abnormalphysiology and development disorders, can also be used as an anti-viralagent Human beta-chemokine Chemokines are a family of related small,secreted proteins involved in biological H1305 (MCP-2) GeneSeq processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.Accession W26655 Members of this family are involved in a similarlydiverse range of pathologies WO9725427 including inflammation, allergy,tissue rejection, viral infection, and tumor biology. TheP80075/CCL8_HUMAN C- chemokines exert their effects by acting on afamily of seven transmembrane G- C motif chemokine 8 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 344) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Chemotaxis, blood-related disorders, viral infection, HIV, woundhealing, cancer Human eosinocyte CC Chemokines are a family of relatedsmall, secreted proteins involved in biological type chemokine eotaxinprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. GeneSeq Accession Members of this family are involved in asimilarly diverse range of pathologies W14990 WO9712914 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The P51671/CCL11_HUMAN chemokines exert their effects by actingon a family of seven transmembrane G- Eotaxin protein coupled receptors.Over 40 human chemokines have been described, which (SEQ ID NO: 245)bind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ.Inflammatory and immune disorders Human thymus and Chemokines are afamily of related small, secreted proteins involved in biologicalactivation regulated processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. cytokine (TARC) GeneSeq Members of thisfamily are involved in a similarly diverse range of pathologiesAccession W14018 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The WO9711969 chemokines exert theireffects by acting on a family of seven transmembrane G-Q92583/CCL17_HUMAN protein coupled receptors. Over 40 human chemokineshave been described, which C-C motif chemokine 17 bind to ~17 receptorsthus far identified. Chemokine activities can be determined (SEQ ID NO:261) using assays known in the art: Methods in molecular Biology, 2000,vol. 138: Chemokine Protocols, Edited by A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power Humana Press Inc., Totowa, NJ Inflammatory andimmune disorders Human chemokine beta-8 Chemokines are a family ofrelated small, secreted proteins involved in biological short formsGeneSeq processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Accession W16315 Members of this family areinvolved in a similarly diverse range of pathologies WO9712041 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The Wildtype chemokine beta- chemokines exert their effects byacting on a family of seven transmembrane G- 8 provided as: proteincoupled receptors. Over 40 human chemokines have been described, whichP55773|CCL23_HUMAN bind to ~17 receptors thus far identified. Chemokineactivities can be determined C-C motif chemokine 23 using assays knownin the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO:459) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing,immune disorders Microphage derived Chemokines are a family of relatedsmall, secreted proteins involved in biological chemokine, MDC processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.GeneSeq Accession Members of this family are involved in a similarlydiverse range of pathologies W20058 WO9640923 including inflammation,allergy, tissue rejection, viral infection, and tumor biology. TheO00626/CCL22_HUMAN chemokines exert their effects by acting on a familyof seven transmembrane G- C-C motif chemokine 22 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 345) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inflammatory diseases, wound healin, angiogenesis Human chemokineZSIG- Chemokines are a family of related small, secreted proteinsinvolved in biological 35 GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W30565 WO9844117Members of this family are involved in a similarly diverse range ofpathologies SEQ ID NO: 2 of WO including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The WO9844117 chemokinesexert their effects by acting on a family of seven transmembrane G- (SEQID NO: 413) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Inflammatory and immune diseases Primate CCchemokine Chemokines are a family of related small, secreted proteinsinvolved in biological “ILINCK” GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. AccesssionW69990 Chemokine activities can be determined using assays known in theart: Methods in WO98328658 Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, SEQ ID NO: 4 from T. N. C.Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. WO9832858 Immuneand inflammatory disorders, abnormal proliferation, regeneration, (SEQID NO: 414) generation and atrophy disorders Primate CXC chemokineChemokines are a family of related small, secreted proteins involved inbiological “IBICK” GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession W69989 Members ofthis family are involved in a similarly diverse range of pathologiesWO9832858 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The SEQ ID NO: 2 from chemokines exerttheir effects by acting on a family of seven transmembrane G- WO9832858protein coupled receptors. Over 40 human chemokines have been described,which (SEQ ID NO: 415) bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune and inflammatory disorders, abnormalproliferation, regeneration, generation and atrophy disorders HumanCC-type Chemokines are a family of related small, secreted proteinsinvolved in biological chemokine protein processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. designated SLCMembers of this family are involved in a similarly diverse range ofpathologies (secondary lymphoid including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The chemokine) GeneSeqchemokines exert their effects by acting on a family of seventransmembrane G- Accession W69163 protein coupled receptors. Over 40human chemokines have been described, which WO9831809 bind to ~17receptors thus far identified. Chemokine activities can be determinedO00585/CCL21_HUMAN using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: C-C motif chemokine 21 Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ IDNO: 346) Humana Press Inc., Totowa, NJ. Immune, inflammatory, andinfectious disorders, cancer Human CC chemokine Chemokines are a familyof related small, secreted proteins involved in biological ELC proteinGeneSeq processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Accession W62542 Members of this family areinvolved in a similarly diverse range of pathologies WO9826071 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The Q99731/CCL19_HUMAN chemokines exert their effects by actingon a family of seven transmembrane G- C-C motif chemokine 19 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 249) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Cancer and infectious diseases, particularly herpes virusHuman DVic-1 C-C Chemokines are a family of related small, secretedproteins involved in biological chemokine GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Accession W60649Members of this family are involved in a similarly diverse range ofpathologies WO9823750 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exerttheir effects by acting on a family of seven transmembrane G- WO9823750protein coupled receptors. Over 40 human chemokines have been described,which (SEQ ID NO: 416) bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Abnormal proliferation, regeneration,degeneration, and atrophy disorders, including cancer Human C-Cchemokine Chemokines are a family of related small, secreted proteinsinvolved in biological DGWCC GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Accession W60650Members of this family are involved in a similarly diverse range ofpathologies WO9823750 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 6 of chemokines exerttheir effects by acting on a family of seven transmembrane G- WO9823750protein coupled receptors. Over 40 human chemokines have been described,which (SEQ ID NO: 417) bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune disorders, cell proliferation disorders,cancer Human STCP-1 GeneSeq Chemokines are a family of related small,secreted proteins involved in biological Accession W62783 processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.WO9824907 Members of this family are involved in a similarly diverserange of pathologies O00626/CCL22_HUMAN including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The C-C motifchemokine 22 chemokines exert their effects by acting on a family ofseven transmembrane G- (SEQ ID NO: 345) protein coupled receptors. Over40 human chemokines have been described, which bind to ~17 receptorsthus far identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders,particularly T cell related disorders, viral infection, andinflammation, especially joint Exodus protein GeneSeq Chemokines are afamily of related small, secreted proteins involved in biologicalAccession W61279 processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. WO9821330 Members of this family are involved ina similarly diverse range of pathologies P78556/CCL20_HUMAN includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The C-C motif chemokine 20 chemokines exert their effects byacting on a family of seven transmembrane G- (isoform 1) protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 248) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune and inflammatory disorders, angiogenesis, cancer, andproliferation disorders, particularly myeloproliferative diseases HumanChr19kine protein Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Acession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W50887 WO9814581Members of this family are involved in a similarly diverse range ofpathologies SEQ ID NO: 10 of including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The WO9814581 chemokinesexert their effects by acting on a family of seven transmembrane G- (SEQID NO: 418) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Cancer and de-generative disorders Human T cellmixed Chemokines are a family of related small, secreted proteinsinvolved in biological lymphocyte reaction processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. expressedchemokine Members of this family are involved in a similarly diverserange of pathologies (TMEC) GeneSeq including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The AccessionW58703 U.S. chemokines exert their effects by acting on a family ofseven transmembrane G- Pat. No. 5,780,268 protein coupled receptors.Over 40 human chemokines have been described, which SEQ ID NO: 2 of U.S.Pat. bind to ~17 receptors thus far identified. Chemokine activities canbe determined No. 5,780,268 using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: (SEQ ID NO: 460) Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Immune, inflammatory, and infectious disorders,cancer Human 6CKine protein Chemokines are a family of related small,secreted proteins involved in biological GeneSeq Accession processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.W50885 WO9814581 Members of this family are involved in a similarlydiverse range of pathologies SEQ ID NO: 8 of including inflammation,allergy, tissue rejection, viral infection, and tumor biology. TheWO9814581 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 419) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Cancer and de-generativedisorders human liver and activation Chemokines are a family of relatedsmall, secreted proteins involved in biological regulated chemokineprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. (LARC) GeneSeq Members of this family are involved in asimilarly diverse range of pathologies Accession W57475 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The WO9817800 chemokines exert their effects by acting on afamily of seven transmembrane G- P78556/CCL20_HUMAN protein coupledreceptors. Over 40 human chemokines have been described, which C-C motifchemokine 20 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (isoform 1) using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 248) ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Immune, inflammatory, andinfectious disorders, cancer RANTES peptide Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. W29538 WO9744462 Members of this family areinvolved in a similarly diverse range of pathologies Wildtype Rantesprovided including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The herien as chemokines exert theireffects by acting on a family of seven transmembrane G-P13501/CCL5_HUMAN C- protein coupled receptors. Over 40 human chemokineshave been described, which C motif chemokine 5 bind to ~17 receptorsthus far identified. Chemokine activities can be determined (SEQ ID NO:241) using assays known in the art: Methods in Molecular Biology, 2000,vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Infectiousdiseases, particularly HIV RANTES 8-68 GeneSeq Chemokines are a familyof related small, secreted proteins involved in biological AccessionW29529 processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. WO9744462 Members of this family are involved in asimilarly diverse range of pathologies Wildtype Rantes providedincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The herien as chemokines exert their effects by acting ona family of seven transmembrane G- P13501/CCL5_HUMAN C- protein coupledreceptors. Over 40 human chemokines have been described, which C motifchemokine 5 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 241) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Infectious diseases, particularly HIV RANTES9-68 GeneSeq Chemokines are a family of related small, secreted proteinsinvolved in biological Accession W29528 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. WO9744462Members of this family are involved in a similarly diverse range ofpathologies Wildtype Rantes provided including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The herien aschemokines exert their effects by acting on a family of seventransmembrane G- P13501/CCL5_HUMAN C- protein coupled receptors. Over 40human chemokines have been described, which C motif chemokine 5 bind to~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 241) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Infectious diseases, particularly HIV Human chemokineprotein Chemokines are a family of related small, secreted proteinsinvolved in biological 331D5 GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Accession W59433Members of this family are involved in a similarly diverse range ofpathologies WO9811226 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 12 of chemokinesexert their effects by acting on a family of seven transmembrane G-WO9811226 protein coupled receptors. Over 40 human chemokines have beendescribed, which (SEQ ID NO: 420) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Abnormal proliferation,regeneration, degeneration, or atrophy, including cancer Human chemokineprotein Chemokines are a family of related small, secreted proteinsinvolved in biological 61164 GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Accession W59430Members of this family are involved in a similarly diverse range ofpathologies WO9811226 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 6 of chemokines exerttheir effects by acting on a family of seven transmembrane G- WO9811226protein coupled receptors. Over 40 human chemokines have been described,which (SEQ ID NO: 421) bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Abnormal proliferation, regeneration,degeneration, or atrophy, including cancer Chemokine MCP-4 Chemokinesare a family of related small, secreted proteins involved in biologicalGeneSeq Accession processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. W56690 WO9809171 Members of this family areinvolved in a similarly diverse range of pathologies Q99616/CCL13_HUMANincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The C-C motif chemokine 13 chemokines exert their effectsby acting on a family of seven transmembrane G- (SEQ ID NO: 347) proteincoupled receptors. Over 40 human chemokines have been described, whichbind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune,Inflammatory, and infectious diseases Human stromal cell- Chemokines area family of related small, secreted proteins involved in biologicalderived chemokine, SDF-1 processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. GeneSeq Accession Members ofthis family are involved in a similarly diverse range of pathologiesW50766 FR2751658 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The P48061/SDF1_HUMAN chemokinesexert their effects by acting on a family of seven transmembrane G-Stromal cell-derived factor 1 protein coupled receptors. Over 40 humanchemokines have been described, which (isoform beta) bind to ~17receptors thus far identified. Chemokine activities can be determined(SEQ ID NO: 260) using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. HIV infections Thymus expressed Chemokines are a family of relatedsmall, secreted proteins involved in biological chemokine (TECK)processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. GeneSeq Accession Members of this family are involved in asimilarly diverse range of pathologies W44397 WO9801557 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The O15444/CCL25_HUMAN chemokines exert their effects by actingon a family of seven transmembrane G- C-C motif chemokine 25 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 348) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Immune and inflammatory disorders Human chemokine MIP-Chemokines are a family of related small, secreted proteins involved inbiological 3alpha GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession W44398 Members ofthis family are involved in a similarly diverse range of pathologiesWO9801557 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P78556/CCL20_HUMAN chemokines exerttheir effects by acting on a family of seven transmembrane G- C-C motifchemokine 20 protein coupled receptors. Over 40 human chemokines havebeen described, which (isoform 1) bind to ~17 receptors thus faridentified. Chemokine activities can be determined (SEQ ID NO: 248)using assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatorydisorders Human chemokine MIP- Chemokines are a family of related small,secreted proteins involved in biological 3beta GeneSeq Accessionprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. W44399 WO9801557 Members of this family are involved in asimilarly diverse range of pathologies Q99731/CCL19_HUMAN includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The C-C motif chemokine 19 chemokines exert their effects byacting on a family of seven transmembrane G- (SEQ ID NO: 249) proteincoupled receptors. Over 40 human chemokines have been described, whichbind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune andinflammatory disorders Human monocyte Chemokines are a family of relatedsmall, secreted proteins involved in biological chemotactic proproteinprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. (MCPP) sequence Members of this family are involved in asimilarly diverse range of pathologies GeneSeq Accession includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The W42072 WO9802459 chemokines exert their effects by actingon a family of seven transmembrane G- SEQ ID NO: 1 of protein coupledreceptors. Over 40 human chemokines have been described, which WO9802459bind to ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 456) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Immune disorders, respiratory disorders, cancerMacrophage-derived Chemokines are a family of related small, secretedproteins involved in biological chemokine (MDC) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccessions Members of this family are involved in a similarly diverserange of pathologies W40811 and Y24414 U.S. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The Pat.No. 5,688,927/U.S. chemokines exert their effects by acting on a familyof seven transmembrane G- Pat. No. 5,932,703 protein coupled receptors.Over 40 human chemokines have been described, which O00626/CCL22_HUMANbind to ~17 receptors thus far identified. Chemokine activities can bedetermined C-C motif chemokine 22 using assays known in the art: Methodsin Molecular Biology, 2000, vol. 138: (SEQ ID NO: 345) ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Immune, and inflammatorydisorders, cancer Macrophage derived Chemokines are a family of relatedsmall, secreted proteins involved in biological chemokine analogueprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. MDC-eyfy GeneSeq Members of this family are involved in asimilarly diverse range of pathologies Accession Y24416 U.S. includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The Pat. No. 5,932,703 (“eyfy” chemokines exert their effectsby acting on a family of seven transmembrane G- disclosed as SEQ ID NO:protein coupled receptors. Over 40 human chemokines have been described,which 546) bind to ~17 receptors thus far identified. Chemokineactivities can be determined Wildtype MDC is SEQ ID using assays knownin the art: Methods in Molecular Biology, 2000, vol. 138: NO: 2 of5,932,703 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power. (SEQ ID NO: 422) Humana Press Inc., Totowa, NJ.Immune and inflammatory disorders Macrophage derived Chemokines are afamily of related small, secreted proteins involved in biologicalchemokine analogue MDC processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. (n + 1) GeneSeq Members of thisfamily are involved in a similarly diverse range of pathologiesAccession Y24413 U.S. including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The Pat. No. 5,932,703 chemokinesexert their effects by acting on a family of seven transmembrane G-protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune and inflammatory disorders Macrophage derived Chemokines area family of related small, secreted proteins involved in biologicalchemokine analogue processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. MDC-yl GeneSeq Members of this family areinvolved in a similarly diverse range of pathologies Accession Y24415U.S. including inflammation, allergy, tissue rejection, viral infection,and tumor biology. The Pat. No. 5,932,703 chemokines exert their effectsby acting on a family of seven transmembrane G- protein coupledreceptors. Over 40 human chemokines have been described, which bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune andinflammatory disorders Human type CC Chemokines are a family of relatedsmall, secreted proteins involved in biological chemokine eotaxin 3processes ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. protein sequence Members of this family are involved in asimilarly diverse range of pathologies GeneSeq Accession includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The Y43178 JP11243960 chemokines exert their effects by actingon a family of seven transmembrane G- Q9Y258/CCL26_HUMAN protein coupledreceptors. Over 40 human chemokines have been described, which C-C motifchemokine 26 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 349) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Allergic diseases and HIV infection Human MCP-3and human Chemokines are a family of related small, secreted proteinsinvolved in biological Muc-1 core epitope (VNT) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. fusion proteinGeneSeq Members of this family are involved in a similarly diverse rangeof pathologies Acession Y29893 including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The WO9946392 chemokinesexert their effects by acting on a family of seven transmembrane G-Wildtype MCP-3 has the protein coupled receptors. Over 40 humanchemokines have been described, which sequence: bind to ~17 receptorsthus far identified. Chemokine activities can be determinedP80098/CCL7_HUMAN C- using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: C motif chemokine 7 Chemokine Protocols. Editedby: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO:336) Humana Press Inc., Totowa, NJ. Cancer and immune disorders,particularly HIV Wildtype Muc-1 has the infection sequence:P15941|MUC1_HUMAN Mucin-1 (isoform 1) (SEQ ID NO: 461) Human IP-10 andhuman Chemokines are a family of related small, secreted proteinsinvolved in biological Muc-1 core epitope (VNT) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. fusion proteinGeneSeq Members of this family are involved in a similarly diverse rangeof pathologies Accession Y29894 including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The WO9946392 chemokinesexert their effects by acting on a family of seven transmembrane G-Wildtype IP10 has the protein coupled receptors. Over 40 humanchemokines have been described, which sequence: bind to ~17 receptorsthus far identified. Chemokine activities can be determinedP02778/CXL10_HUMAN using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: C—X—C motif chemokine 10 Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ IDNO: 242) Humana Press Inc., Totowa, NJ. Cancer and immune disorders,particularly HIV Wildtype Muc-1 has the infection sequence:P15941|MUC1_HUMAN Mucin-1 (isoform 1) (SEQ ID NO: 461) Human IP-10 andHIV-1 Chemokines are a family of related small, secreted proteinsinvolved in biological gp 120 hyper-variable processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. region fusionprotein Members of this family are involved in a similarly diverse rangeof pathologies GeneSeq Accession including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The Y29897 W09946392chemokines exert their effects by acting on a family of seventransmembrane G- Wildtype IP10 has the protein coupled receptors. Over40 human chemokines have been described, which sequence: bind to ~17receptors thus far identified. Chemokine activities can be determinedP02778/CXL10_HUMAN using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: C—X—C motif chemokine 10 Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ IDNO: 242) Humana Press Inc., Totowa, NJ. Cancer and immune disorders,particularly HIV Wildtype gp120 has the infection sequence:P03378|32-509 (cleaved product of gp160) (SEQ ID NO: 462) Human mammaryChemokines are a family of related small, secreted proteins involved inbiological associated chemokine processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. (MACK) protein Full- Members ofthis family are involved in a similarly diverse range of pathologiesLength and Mature including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The GeneSeq Accessions chemokinesexert their effects by acting on a family of seven transmembrane G-Y29092 and Y29093 protein coupled receptors. Over 40 human chemokineshave been described, which WO9936540 bind to ~17 receptors thus faridentified. Chemokine activities can be determined Full-length: SEQ IDNO: 1 using assays known in the art: Methods in Molecular Biology, 2000,vol. 138: of WO9936540 Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 423) HumanaPress Inc., Totowa, NJ. Breast disease, including cancer Mature Form:SEQ ID NO: 2 of WO9936540 (SEQ ID NO: 424) Tim-1 protein GeneSeqChemokines are a family of related small, secreted proteins involved inbiological Accession Y28290 processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. WO9933990 Members of thisfamily are involved in a similarly diverse range of pathologies SEQ IDNO: 2 of including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The WO9933990 chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 350)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inflammation due to stimuli such as heart attacks and stroke,infection, physical trauma, UV or ionizing radiation, burns, frostbiteor corrosive chemicals Human Lkn-1 Full-Length Chemokines are a familyof related small, secreted proteins involved in biological and Matureprotein processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. GeneSeq Accessions Members of this family areinvolved in a similarly diverse range of pathologies Y17280, Y17274,Y17281, including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The and Y17275 WO9928473 chemokines exerttheir effects by acting on a family of seven transmembrane G- andWO9928472 protein coupled receptors. Over 40 human chemokines have beendescribed, which Q16663/CCL15_HUMAN bind to ~17 receptors thus faridentified. Chemokine activities can be determined C-C motif chemokine15 using assays known in the art: Methods in Molecular Biology, 2000,vol. 138: (SEQ ID NO: 342) Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. HIV infection and cancer, particularly leukemia N-terminal modifiedChemokines are a family of related small, secreted proteins involved inbiological chemokine met-hSDF-1 processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. alpha GeneSeq Accession Membersof this family are involved in a similarly diverse range of pathologiesY05818 WO9920759 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 10 of chemokinesexert their effects by acting on a family of seven transmembrane G-WO9920759 protein coupled receptors. Over 40 human chemokines have beendescribed, which (SEQ ID NO: 425) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Inhibit or stimulate angiogenesis,inhibit the binding of HIV N-terminal modified Chemokines are a familyof related small, secreted proteins involved in biological chemokinemet-hSDF-1 processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. beta GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies Y05819 WO9920759including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 11 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9920759 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 426) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Inhibit or stimulate angiogenesis, inhibit the binding of HIV,antiinflammatory; immunosuppressant N-terminal modified Chemokines are afamily of related small, secreted proteins involved in biologicalchemokine processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. GroHEK/hSDF-1alpha Members of this family areinvolved in a similarly diverse range of pathologies GeneSeq Accessionincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The Y05820 WO9920759 chemokines exert their effects byacting on a family of seven transmembrane G- SEQ ID NO: 12 of proteincoupled receptors. Over 40 human chemokines have been described, whichWO9920759 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 427) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Inhibit or stimulate angiogenesis, inhibit thebinding of HIV, antiinflammatory; immunosuppressant N-terminal modifiedChemokines are a family of related small, secreted proteins involved inbiological chemokine processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. GroHEK/hSDF-1beta. Members of this family areinvolved in a similarly diverse range of pathologies GeneSeq Accessionincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The Y05821 WO9920759 chemokines exert their effects byacting on a family of seven transmembrane G- SEQ ID NO: 13 of proteincoupled receptors. Over 40 human chemokines have been described, whichWO9920759 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 428) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Inhibit or stimulate angiogenesis, inhibit thebinding of HIV, antiinflammatory; immunosuppressant Chemokine EotaxinChemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Y14230 WO9912968 Members ofthis family are involved in a similarly diverse range of pathologiesP51671/CCL11_HUMAN including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The Eotaxin chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 245)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Increase or enhance an inflammatory response, an immune responseorhaematopoietic cell-associated activity; treat a vascular indication;Cancer; enhance wound healing, to prevent or treat asthma, organtransplant rejction, rheumatoid arthritis or allergy Chemokine hMCP1aChemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Y14225 WO9912968 Members ofthis family are involved in a similarly diverse range of pathologiesincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The chemokines exert their effects by acting on a familyof seven transmembrane G- protein coupled receptors. Over 40 humanchemokines have been described, which bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vasculardisorders, Wound healing, cancer, prevent organ transplant rejection,Increase or enhance an inflammatory response, Chemokine hMCP1bChemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Y14226 WO9912968 Members ofthis family are involved in a similarly diverse range of pathologiesincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The chemokines exert their effects by acting on a familyof seven transmembrane G- protein coupled receptors. Over 40 humanchemokines have been described, which bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vasculardisorders, Wound healing, cancer, prevent organ transplant rejection,Increase or enhance an inflammatory response, Chemokine hSDF1bChemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Y14228 WO9912968 Members ofthis family are involved in a similarly diverse range of pathologiesP48061/SDF1_HUMAN including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The Stromal cell-derived factor 1chemokines exert their effects by acting on a family of seventransmembrane G- (isoform beta) protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 260) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders,Vascular disorders, Wound healing, cancer, prevent organ transplantrejection, Increase or enhance an inflammatory response, Chemokine hIL-8Chemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Y14229 WO9912968 Members ofthis family are involved in a similarly diverse range of pathologiesP10145/IL8_HUMAN including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The Interleukin-8 chemokines exerttheir effects by acting on a family of seven transmembrane G-(isoform 1) protein coupled receptors. Over 40 human chemokines havebeen described, which (SEQ ID NO: 341) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science253, 1278-80. Immune disorders, Vascular disorders, Wound healing,cancer, prevent organ transplant rejection, Increase or enhance aninflammatory response, Chemokine hMCP1 Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Y14222 WO9912968 Members of this family areinvolved in a similarly diverse range of pathologies P13500/CCL2_HUMANC- including inflammation, allergy, tissue rejection, viral infection,and tumor biology. The C motif chemokine 2 chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 337)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune disorders, Vascular disorders, Wound healing, cancer, preventorgan transplant rejection, Increase or enhance an inflammatoryresponse, Chemokine hMCP2 Chemokines are a family of related small,secreted proteins involved in biological GeneSeq Accession processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.Y14223 WO9912968 Members of this family are involved in a similarlydiverse range of pathologies P80075/CCL8_HUMAN C- includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The C motif chemokine 8 chemokines exert their effects byacting on a family of seven transmembrane G- (SEQ ID NO: 344) proteincoupled receptors. Over 40 human chemokines have been described, whichbind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders, Vascular disorders, Wound healing, cancer, prevent organtransplant rejection, Increase or enhance an inflammatory response,Chemokine hMCP3 Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Y14224 WO9912968Members of this family are involved in a similarly diverse range ofpathologies P80098/CCL7_HUMAN C- including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C motif chemokine 7chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 336) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vasculardisorders, Wound healing, cancer, prevent organ transplant rejection,Increase or enhance an inflammatory response, C-C chemokine, MCP2Chemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Y05300 EP905240 Members of thisfamily are involved in a similarly diverse range of pathologiesP80075/CCL8_HUMAN C- including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The C motif chemokine 8 chemokinesexert their effects by acting on a family of seven transmembrane G- (SEQID NO: 344) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Inflammatory, Immune and infectious diseases;pulmonary diseases and skin disorders; tumours, and angiogenesis-andhaematopoiesis-related diseases Wild type monocyte Chemokines are afamily of related small, secreted proteins involved in biologicalchemotactic protein 2 processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. GeneSeq Accession Members ofthis family are involved in a similarly diverse range of pathologiesY07233 EP906954 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P80075/CCL8_HUMAN C- chemokines exerttheir effects by acting on a family of seven transmembrane G- C motifchemokine 8 protein coupled receptors. Over 40 human chemokines havebeen described, which (SEQ ID NO: 344) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Inflammatory, Immune andinfectious diseases; pulmonary diseases and skin disorders; tumours, andangiogenesis-and haematopoiesis-related diseases Truncated monocyteChemokines are a family of related small, secreted proteins involved inbiological chemotactic protein 2 (6- processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. 76) GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies Y07234 EP906954 including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The FIG. 1 ofEP905241 and chemokines exert their effects by acting on a family ofseven transmembrane G- EP906954 protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 429) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power, Humana Press Inc., Totowa, NJ Inflammatory, Immune andinfectious diseases; pulmonary diseases and skin disorders; tumours, andangiogenesis-and haematopoiesis-related diseases Truncated RANTESChemokines are a family of related small, secreted proteins involved inbiological protein (3-68) GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accessions Y07236 and Membersof this family are involved in a similarly diverse range of pathologiesY07232 EP905241; including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The EP906954 chemokines exert theireffects by acting on a family of seven transmembrane G- FIG. 1 ofEP906954 protein coupled receptors. Over 40 human chemokines have beendescribed, which (SEQ ID NO: 430) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power, Humana Press Inc., Totowa, NJ Inflammatory, immune and infectiousdiseases; pulmonary diseases and skin disorders; tumours, andangiogenesis-and haematopoiesis-related diseases Wild type monocyteChemokines are a family of related small, secreted proteins involved inbiological chemotactic protein 2 processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. GeneSeq Accession Members ofthis family are involved in a similarly diverse range of pathologiesY07237 EP905241 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P80075/CCL8_HUMAN C- chemokines exerttheir effects by acting on a family of seven transmembrane G- C motifchemokine 8 protein coupled receptors. Over 40 human chemokines havebeen described, which (SEQ ID NO: 344) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power, Humana Press Inc., Totowa, NJ Inflammatory, immune and infectiousdiseases; pulmonary diseases and skin disorders; tumours, andangiogenesis-and haematopoiesis-related diseases Truncated monocyteChemokines are a family of related small, secreted proteins involved inbiological chemotactic protein 2 (6- processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. 76) GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies Y07238 EP905241 including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The FIG. 1 ofEP905241 and chemokines exert their effects by acting on a family ofseven transmembrane G- EP906954 protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 429) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power, Humana Press Inc., Totowa, NJ Inflammatory, immune andinfectious diseases; pulmonary diseases and skin disorders; tumours, andangiogenesis-and haematopoiesis-related diseases A partial CXCR4Bprotein Chemokines are a family of related small, secreted proteinsinvolved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W97363 EP897980Members of this family are involved in a similarly diverse range ofpathologies SEQ ID NO: 2 of including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The EP897980 chemokinesexert their effects by acting on a family of seven transmembrane G- (sEQID NO: 431) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, HumanaPress Inc., Totowa, NJ Soluble CXCR4B receptor polypeptides may beuseful for inhibiting chemokine activities and viral infection.Interferon gamma- Chemokines are a family of related small, secretedproteins involved in biological inducible protein (IP-10) processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.GeneSeq Accession Members of this family are involved in a similarlydiverse range of pathologies W96709 U.S. Pat. No. includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The 5,871,723 chemokines exert their effects by acting on afamily of seven transmembrane G- P02778/CXL10_HUMAN protein coupledreceptors. Over 40 human chemokines have been described, which C—X—Cmotif chemokine 10 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 242) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, HumanaPress Inc., Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immunedisorders, Cardio-Vascular disorders, Musco-skeletal disorders Amonokine induced by Chemokines are a family of related small, secretedproteins involved in biological gamma-interferon (MIG) processes rangingfrom hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96710 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- Q07325/CXCL9_HUMAN protein coupled receptors. Over 40human chemokines have been described, which C—X—C motif chemokine 9 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 351) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc.,Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders Interleukin-8 (IL-8)protein. Chemokines are a family of related small, secreted proteinsinvolved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. W96711 U.S. Pat.No. Members of this family are involved in a similarly diverse range ofpathologies 5,871,723 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The P10145/IL8_HUMAN chemokinesexert their effects by acting on a family of seven transmembrane G-Interleukin-8 protein coupled receptors. Over 40 human chemokines havebeen described, which (isoform 1) bind to ~17 receptors thus faridentified. Chemokine activities can be determined (SEQ ID NO: 341)using assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991)Science 253, 1278-80. Angiogenesis, Cancer, Inflammatory and Immunedisorders, Cardio-Vascular disorders, Musco-skeletal disordersEpithelial neutrophil Chemokines are a family of related small, secretedproteins involved in biological activating protein-78 processes rangingfrom hematopoiesis, angiogenesis, and leukocyte trafficking. (ENA-78)GeneSeq Members of this family are involved in a similarly diverse rangeof pathologies Accession W96712 U.S. including inflammation, allergy,tissue rejection, viral infection, and tumor biology. The Pat. No.5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- P42830/CXCL5_HUMAN protein coupled receptors. Over 40human chemokines have been described, which C—X—C motif chemokine 5 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 352) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc.,Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders Growth relatedoncogene- Chemokines are a family of related small, secreted proteinsinvolved in biological alpha (GRO-alpha). processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96713 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- P09341/GROA_HUMAN protein coupled receptors. Over 40human chemokines have been described, which Growth-regulated alpha bindto ~17 receptors thus far identified. Chemokine activities can bedetermined protein using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: (SEQ ID NO: 340) Chemokine Protocols. Editedby: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana PressInc., Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immunedisorders, Cardio-Vascular disorders, Musco-skeletal disorders Growthrelated oncogene- Chemokines are a family of related small, secretedproteins involved in biological beta (GRO-beta). processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96714 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- P19875/CXCL2_HUMAN protein coupled receptors. Over 40human chemokines have been described, which C—X—C motif chemokine 2 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 338) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders Growth relatedoncogene- Chemokines are a family of related small, secreted proteinsinvolved in biological gamma (GRO-gamma) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96715 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- P19876/CXCL3_HUMAN protein coupled receptors. Over 40human chemokines have been described, which C—X—C motif chemokine 3 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 339) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders A platelet basicprotein Chemokines are a family of related small, secreted proteinsinvolved in biological (PBP) GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Accession W96716U.S. Members of this family are involved in a similarly diverse range ofpathologies Pat. No. 5,871,723 including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The P02775/CXCL7_HUMANchemokines exert their effects by acting on a family of seventransmembrane G- Platelet basic protein protein coupled receptors. Over40 human chemokines have been described, which (SEQ ID NO: 353) bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ.Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio-Vasculardisorders, Musco-skeletal disorders Connective tissue Chemokines are afamily of related small, secreted proteins involved in biologicalactivating protein-III processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. (CTAP-III) GeneSeqAccessionMembers of this family are involved in a similarly diverse range ofpathologies S96717 U.S. Pat. including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The No. 5,871,723chemokines exert their effects by acting on a family of seventransmembrane G- SEQ ID NO: 9 of U.S. protein coupled receptors. Over 40human chemokines have been described, which Pat. No. 5,871,723 bind to~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 354) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disordersBeta-thrombo-globulin Chemokines are a family of related small, secretedproteins involved in biological protein (beta-TG) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96718 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- SEQ ID NO: 10 of U.S. protein coupled receptors. Over40 human chemokines have been described, which Pat. No. 5,871,723 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 355) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders Neutrophilactivating Chemokines are a family of related small, secreted proteinsinvolved in biological peptide-2 (NAP-2) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96719 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- SEQ ID NO: 11 of U.S. protein coupled receptors. Over40 human chemokines have been described, which Pat. No. 5,871,723 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 356) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders Granulocytechemotactic Chemokines are a family of related small, secreted proteinsinvolved in biological protein-2 (GCP-2) processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies W96720 U.S. Pat. No. including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The5,871,723 chemokines exert their effects by acting on a family of seventransmembrane G- P80162/CXCL6_HUMAN protein coupled receptors. Over 40human chemokines have been described, which C—X—C motif chemokine 6 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 357) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders,Cardio-Vascular disorders, Musco-skeletal disorders Human chemokine MIG-Chemokines are a family of related small, secreted proteins involved inbiological beta protein GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession W90124 Members ofthis family are involved in a similarly diverse range of pathologiesEP887409 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The (SEQ ID NO: 463) chemokines exerttheir effects by acting on a family of seven transmembrane G- proteincoupled receptors. Over 40 human chemokines have been described, whichbind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders, viral, parasitic, fungal or bacterial infections, Cancer;autoimmune diseases or transplant rejection Human ZCHEMO-8 Chemokinesare a family of related small, secreted proteins involved in biologicalGeneSeq Accession processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. W82716 WO9854326 Members of this family areinvolved in a similarly diverse range of pathologies SEQ ID NO: 2 ofincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The WO9854326 chemokines exert their effects by acting ona family of seven transmembrane G- (SEQ ID NO: 432) protein coupledreceptors. Over 40 human chemokines have been described, which bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders, cancer, myelopoietic disorders, autoimmune disorders andimmunodeficiencies, Inflammatory and infectious diseases, Vasculardisorders, wound healing Human Act-2 protein Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. W82717 WO9854326 Members of this family areinvolved in a similarly diverse range of pathologies P13236/CCL4_HUMANC- including inflammation, allergy, tissue rejection, viral infection,and tumor biology. The C motif chemokine 4 chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 358)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune disorders, cancer, myelopoietic disorders, autoimmunedisorders and immunodeficiencies, Inflammatory and infectious diseases,Vascular disorders, wound healing Human SISD protein Chemokines are afamily of related small, secreted proteins involved in biologicalGeneSeq Acession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. W82720 WO9854326 Members of this family areinvolved in a similarly diverse range of pathologies P13501/CCL5_HUMANC- including inflammation, allergy, tissue rejection, viral infection,and tumor biology. The C motif chemokine 5 chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 241)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune disorders, cancer, myelopoietic disorders, autoimmunedisorders and immunodeficiencies, Inflammatory and infectious diseases,Vascular disorders, wound healing Human MI10 protein Chemokines are afamily of related small, secreted proteins involved in biologicalGeneSeq Accession processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. W82721 WO9854326 Members of this family areinvolved in a similarly diverse range of pathologies SEQ ID NO: 37 ofincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The WO9854326 chemokines exert their effects by acting ona family of seven transmembrane G- (SEQ ID NO: 433) protein coupledreceptors. Over 40 human chemokines have been described, which bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders, cancer, myelopoietic disorders, autoimmune disorders andimmunodeficiencies, Inflammatory and infectious diseases, Vasculardisorders, wound healing Human MI1A protein Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. W82722 W09854326 Members of this family areinvolved in a similarly diverse range of pathologies SEQ ID NO: 38 ofincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The WO9854326 chemokines exert their effects by acting ona family of seven transmembrane G- (SEQ ID NO: 434) protein coupledreceptors. Over 40 human chemokines have been described, which bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders, cancer, myelopoietic disorders, autoimmune disorders andimmunodeficiencies, Inflammatory and infectious diseases, Vasculardisorders, wound healing Human CCC3 protein Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. W82723 WO9854326 Members of this family areinvolved in a similarly diverse range of pathologies SEQ ID NO: 39 ofincluding inflammation, allergy, tissue rejection, viral infection, andtumor biology. The WO9854326 chemokines exert their effects by acting ona family of seven transmembrane G- (SEQ ID NO: 435) protein coupledreceptors. Over 40 human chemokines have been described, which bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immunedisorders, cancer, myelopoietic disorders, autoimmune disorders andimmunodeficiencies, Inflammatory and infectious diseases, Vasculardisorders, wound healing A human L105 chemokine Chemokines are a familyof related small, secreted proteins involved in biological designatedhuL105_3. processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies W87588 WO9856818including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 2 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9856818 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 436) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Cancer, wound healing A human L105 chemokine Chemokines are a familyof related small, secreted proteins involved in biological designatedhuL105_7. processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies W87589 WO9856818including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The SEQ ID NO: 4 of chemokines exert their effects byacting on a family of seven transmembrane G- WO9856818 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 437) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Cancer, wound healing Human mature gro-alpha Chemokines are a familyof related small, secreted proteins involved in biological polypeptideused to treat processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. sepsis GeneSeq Members of this family areinvolved in a similarly diverse range of pathologies Accession W81498including inflammation, allergy, tissue rejection, viral infection, andtumor biology. The WO9848828 chemokines exert their effects by acting ona family of seven transmembrane G- P09341/GROA_HUMAN protein coupledreceptors. Over 40 human chemokines have been described, whichGrowth-regulated alpha bind to ~17 receptors thus far identified.Chemokine activities can be determined protein using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 340)Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Infectious diseases, sepsisHuman mature gro- Chemokines are a family of related small, secretedproteins involved in biological gamma polypeptide used processes rangingfrom hematopoiesis, angiogenesis, and leukocyte trafficking. to treatsepsis GeneSeq Members of this family are involved in a similarlydiverse range of pathologies Accession W81500 including inflammation,allergy, tissue rejection, viral infection, and tumor biology. TheWO9848828 chemokines exert their effects by acting on a family of seventransmembrane G- P19876/CXCL3_HUMAN protein coupled receptors. Over 40human chemokines have been described, which C—X—C motif chemokine 3 bindto ~17 receptors thus far identified. Chemokine activities can bedetermined (SEQ ID NO: 339) using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Infectious diseases, sepsis Human thymus expressedChemokines are a family of related small, secreted proteins involved inbiological chemokine TECK and processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. TECK variant GeneSeq Members ofthis family are involved in a similarly diverse range of pathologiesAccessions B19607 and including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The B19608 WO0053635 chemokinesexert their effects by acting on a family of seven transmembrane G-Wildtype TECK provided protein coupled receptors. Over 40 humanchemokines have been described, which as: bind to ~17 receptors thus faridentified. Chemokine activities can be determined O15444/CCL25_HUMANusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: C-C motif chemokine 25 Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 348) HumanaPress Inc., Totowa, NJ. Inflammatory disorders, cancer, Immune andvascular disorders Human chemokine Chemokines are a family of relatedsmall, secreted proteins involved in biological SDF1alpha GeneSeqprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. Accession B15791 Members of this family are involved in asimilarly diverse range of pathologies WO0042071 including inflammation,allergy, tissue rejection, viral infection, and tumor biology. TheP48061-2/SDF1_HUMAN chemokines exert their effects by acting on a familyof seven transmembrane G- Isoform Alpha of Stromal protein coupledreceptors. Over 40 human chemokines have been described, whichcell-derived factor 1 bind to ~17 receptors thus far identified.Chemokine activities can be determined (isoform alpha) using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ IDNO: 259) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmunedisorders, Immune, Vascular and Inflammatory disorders Human chemokineGRO- Chemokines are a family of related small, secreted proteinsinvolved in biological alpha GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. B15793 WO0042071Members of this family are involved in a similarly diverse range ofpathologies P09341/GROA_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The Growth-regulatedalpha chemokines exert their effects by acting on a family of seventransmembrane G- protein protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 340) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders,Immune, Vascular and Inflammatory disorders Human chemokine eotaxinChemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. B15794 WO0042071 Members ofthis family are involved in a similarly diverse range of pathologiesP51671/CCL11_HUMAN including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The Eotaxin chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 245)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disordersHuman chemokine MIG Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. B15803 WO0042071Members of this family are involved in a similarly diverse range ofpathologies Q07325/CXCL9_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C—X—C motif chemokine9 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 351) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders,Immune, Vascular and Inflammatory disorders Human chemokine PF4Chemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. B15804 WO0042071 Members ofthis family are involved in a similarly diverse range of pathologiesP02776/PLF4_HUMAN including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The Platelet factor 4 chemokinesexert their effects by acting on a family of seven transmembrane G- (SEQID NO: 359) protein coupled receptors. Over 40 human chemokines havebeen described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular andInflammatory disorders Human chemokine I-309 Chemokines are a family ofrelated small, secreted proteins involved in biological GeneSeqAccession processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. B15805 WO0042071 Members of this family areinvolved in a similarly diverse range of pathologies P22362/CCL1_HUMANC- including inflammation, allergy, tissue rejection, viral infection,and tumor biology. The C motif chemokine 1 chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 360)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disordersHuman chemokine HCC-1 Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. B15806 WO0042071Members of this family are involved in a similarly diverse range ofpathologies Q16627/CCL14_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C-C motif chemokine14 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 361) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders,Immune, Vascular and Inflammatory disorders Human chemokine C10Chemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. B15807 WO0042071 Members ofthis family are involved in a similarly diverse range of pathologies SEQID NO: 49 of including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The WO0042071 chemokines exert theireffects by acting on a family of seven transmembrane G- (SEQ ID NO: 438)protein coupled receptors. Over 40 human chemokines have been described,which bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disordersHuman chemokine CCR-2 Chemokines are a family of related small, secretedproteins involved in biological GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. B15808 WO0042071Members of this family are involved in a similarly diverse range ofpathologies P41597/CCR2_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C-C chemokinereceptor chemokines exert their effects by acting on a family of seventransmembrane G- type 2 protein coupled receptors. Over 40 humanchemokines have been described, which (isoform A) bind to ~17 receptorsthus far identified. Chemokine activities can be determined (SEQ ID NO:362) using assays known in the art: Methods in Molecular Biology, 2000,vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C.Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmunedisorders, Immune, Vascular and Inflammatory disorders Human chemokineENA- Chemokines are a family of related small, secreted proteinsinvolved in biological 78 GeneSeq Accession processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. B15809 WO0042071Members of this family are involved in a similarly diverse range ofpathologies P42830/CXCL5_HUMAN including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The C—X—C motif chemokine5 chemokines exert their effects by acting on a family of seventransmembrane G- (SEQ ID NO: 352) protein coupled receptors. Over 40human chemokines have been described, which bind to ~17 receptors thusfar identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders,Immune, Vascular and Inflammatory disorders Human chemokine Chemokinesare a family of related small, secreted proteins involved in biologicalGRObeta GeneSeq processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Accession B15810 Members of this family areinvolved in a similarly diverse range of pathologies WO0042071 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The P19875/CXCL2_HUMAN chemokines exert their effects by actingon a family of seven transmembrane G- C—X—C motif chemokine 2 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 338) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatorydisorders Human chemokine IP-10 Chemokines are a family of relatedsmall, secreted proteins involved in biological GeneSeq Accessionprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. B15811 WO0042071 Members of this family are involved in asimilarly diverse range of pathologies P02778/CXL10_HUMAN includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The C—X—C motif chemokine 10 chemokines exert their effects byacting on a family of seven transmembrane G- (SEQ ID NO: 242) proteincoupled receptors. Over 40 human chemokines have been described, whichbind to ~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmunedisorders, Immune, Vascular and Inflammatory disorders Human chemokineChemokines are a family of related small, secreted proteins involved inbiological SDF1beta GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession B15812 Members ofthis family are involved in a similarly diverse range of pathologiesWO0042071 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P48061/SDF1_HUMAN chemokines exerttheir effects by acting on a family of seven transmembrane G- Stromalcell-derived factor 1 protein coupled receptors. Over 40 humanchemokines have been described, which (isoform beta) bind to ~17receptors thus far identified. Chemokine activities can be determined(SEQ ID NO: 260) using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disordersHuman chemokine GRO Chemokines are a family of related small, secretedproteins involved in biological alpha GeneSeq Accession processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.B15813 WO0042071 Members of this family are involved in a similarlydiverse range of pathologies P09341/GROA_HUMAN including inflammation,allergy, tissue rejection, viral infection, and tumor biology. TheGrowth-regulated alpha chemokines exert their effects by acting on afamily of seven transmembrane G- protein protein coupled receptors. Over40 human chemokines have been described, which (SEQ ID NO: 340) bind to~17 receptors thus far identified. Chemokine activities can bedetermined using assays known in the art: Methods in Molecular Biology,2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T.N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmunedisorders, Immune, Vascular and Inflammatory disorders Human chemokineChemokines are a family of related small, secreted proteins involved inbiological MIP1beta GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession B15831 Members ofthis family are involved in a similarly diverse range of pathologiesWO0042071 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P13236/CCL4_HUMAN C- chemokines exerttheir effects by acting on a family of seven transmembrane G- C motifchemokine 4 protein coupled receptors. Over 40 human chemokines havebeen described, which (SEQ ID NO: 358) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune,Vascular and Inflammatory disorders A human C-C chemokine Chemokines area family of related small, secreted proteins involved in biologicaldesignated exodus processes ranging from hematopoiesis, angiogenesis,and leukocyte trafficking. GeneSeq Accession Members of this family areinvolved in a similarly diverse range of pathologies B07939 U.S. Pat.No. including inflammation, allergy, tissue rejection, viral infection,and tumor biology. The 6,096,300 chemokines exert their effects byacting on a family of seven transmembrane G- P78556/CCL20_HUMAN proteincoupled receptors. Over 40 human chemokines have been described, whichC-C motif chemokine 20 bind to ~17 receptors thus far identified.Chemokine activities can be determined (isoform 1) using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 248)Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Cancer Human chemokineChemokines are a family of related small, secreted proteins involved inbiological L105_7 GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession Y96922 U.S. Membersof this family are involved in a similarly diverse range of pathologiesPat. No. 6,084,071 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The SEQ ID NO: 4 of chemokines exerttheir effects by acting on a family of seven transmembrane G- WO9856818protein coupled receptors. Over 40 human chemokines have been described,which (SEQ ID NO: 437) bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Chemotaxis, Gene Therapy, Wound healing Humanchemokine Chemokines are a family of related small, secreted proteinsinvolved in biological L105_3 GeneSeq processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96923U.S. Members of this family are involved in a similarly diverse range ofpathologies Pat. No. 6,084,071 including inflammation, allergy, tissuerejection, viral infection, and tumor biology. The SEQ ID NO: 2 ofchemokines exert their effects by acting on a family of seventransmembrane G- WO9856818 protein coupled receptors. Over 40 humanchemokines have been described, which (SEQ ID NO: 436) bind to ~17receptors thus far identified. Chemokine activities can be determinedusing assays known in the art: Methods in Molecular Biology, 2000, vol.138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells,and C. A. Power. Humana Press Inc., Totowa, NJ. Chemotaxis, GeneTherapy, Wound healing Human secondary Chemokines are a family ofrelated small, secreted proteins involved in biological lymphoidchemokine processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. (SLC) GeneSeq Members of this family are involvedin a similarly diverse range of pathologies Accession B01434 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The WO0038706 chemokines exert their effects by acting on afamily of seven transmembrane G- O00585/CCL21_HUMAN protein coupledreceptors. Over 40 human chemokines have been described, which C-C motifchemokine 21 bind to ~17 receptors thus far identified. Chemokineactivities can be determined (SEQ ID NO: 346) using assays known in theart: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Cancer, Vascular and Immune disorders Humannon-ELR CXC Chemokines are a family of related small, secreted proteinsinvolved in biological chemokine H174 processes ranging fromhematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeqAccession Members of this family are involved in a similarly diverserange of pathologies Y96310 WO0029439 including inflammation, allergy,tissue rejection, viral infection, and tumor biology. TheO14625/CXL11_HUMAN chemokines exert their effects by acting on a familyof seven transmembrane G- C—X—C motif chemokine 11 protein coupledreceptors. Over 40 human chemokines have been described, which (SEQ IDNO: 363) bind to ~17 receptors thus far identified. Chemokine activitiescan be determined using assays known in the art: Methods in MolecularBiology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa,NJ. Immune and Inflammatory disorders, Cancer, Haemostatic andthrombolytic activity Human non-ELR CXC Chemokines are a family ofrelated small, secreted proteins involved in biological chemokine IP10GeneSeq processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Accession Y96311 Members of this family areinvolved in a similarly diverse range of pathologies WO0029439 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The P02778/CXL10_HUMAN chemokines exert their effects by actingon a family of seven transmembrane G- C—X—C motif chemokine 10 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 242) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Immune and Inflammatory disorders, Cancer, haemostatic andthrombolytic activity Human non-ELR CXC Chemokines are a family ofrelated small, secreted proteins involved in biological chemokine MigGeneSeq processes ranging from hematopoiesis, angiogenesis, andleukocyte trafficking. Accession Y96313 Members of this family areinvolved in a similarly diverse range of pathologies WO0029439 includinginflammation, allergy, tissue rejection, viral infection, and tumorbiology. The Q07325/CXCL9_HUMAN chemokines exert their effects by actingon a family of seven transmembrane G- C—X—C motif chemokine 9 proteincoupled receptors. Over 40 human chemokines have been described, which(SEQ ID NO: 351) bind to ~17 receptors thus far identified. Chemokineactivities can be determined using assays known in the art: Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc.,Totowa, NJ. Immune and Inflammatory disorders, Cancer, haemostatic andthrombolytic activity Human chemokine Chemokines are a family of relatedsmall, secreted proteins involved in biological Ckbeta-7 GeneSeqprocesses ranging from hematopoiesis, angiogenesis, and leukocytetrafficking. Accession Y96280 Members of this family are involved in asimilarly diverse range of pathologies WO0028035 including inflammation,allergy, tissue rejection, viral infection, and tumor biology. The FIG.1 of WO0028035 chemokines exert their effects by acting on a family ofseven transmembrane G- (SEQ ID NO: 439) protein coupled receptors. Over40 human chemokines have been described, which bind to ~17 receptorsthus far identified. Chemokine activities can be determined using assaysknown in the art: Methods in Molecular Biology, 2000, vol. 138:Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, andC. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing,inflammatory and immunoregulatory disorders Human chemokine MIP-Chemokines are a family of related small, secreted proteins involved inbiological 1alpha GeneSeq processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. Accession Y96281 Members ofthis family are involved in a similarly diverse range of pathologiesWO0028035 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The P10147/CCL3_HUMAN C- chemokines exerttheir effects by acting on a family of seven transmembrane G- C motifchemokine 3 protein coupled receptors. Over 40 human chemokines havebeen described, which (SEQ ID NO: 364) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing,inflammatory and immunoregulatory disorders Human mature chemokineChemokines are a family of related small, secreted proteins involved inbiological Ckbeta-7 (optionally processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. truncated) GenSeq Members ofthis family are involved in a similarly diverse range of pathologiesAccession Y96282 including inflammation, allergy, tissue rejection,viral infection, and tumor biology. The WO0028035 chemokines exert theireffects by acting on a family of seven transmembrane G- FIG. 1 ofWO0028035 protein coupled receptors. Over 40 human chemokines have beendescribed, which (SEQ ID NO: 440) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing,inflammatory and immunoregulatory disorders Human chemokine Chemokinesare a family of related small, secreted proteins involved in biologicalreceptor CXCR3 GeneSeq processes ranging from Chemokine activities canbe determined using assays known Accession Y79372 in the art: Methods inMolecular Biology, 2000, vol. 138: Soluble CXCR3 polypeptides WO0018431may be useful for inhibiting P49682|CXCR3_HUMAN C—X—C chemokine receptortype 3 (isoform 1) (SEQ ID NO: 240) Human neurotactin hematopoiesis,angiogenesis, and leukocyte trafficking. Members of this family arechemokine like domain involved in a similarly diverse range ofpathologies including inflammation, allergy, GeneSeq Accession tissuerejection, viral infection, and tumor biology. The chemokines exerttheir effects Y53259 U.S. Pat. No. by acting on a family of seventransmembrane G-protein coupled receptors. Over 40 6,043,086 humanchemokines have been described, which bind to ~17 receptors thus farP78423/X3CL1_HUMAN identified. Chemokine Protocols. Edited by: A. E. I.Proudfoot, T. N. C. Wells, and C. A. Power. Fractalkine Humana PressInc., Totowa, NJ. chemokine activities and viral infection. (SEQ ID NO:244) Human CC type Chemokines are a family of related small, secretedproteins involved in biological chemokine interleukin C processesranging from hematopoiesis, angiogenesis, and leukocyte trafficking.GeneSeq Accession Members of this family are involved in a similarlydiverse range of pathologies Y57771 JP11302298 including inflammation,allergy, tissue rejection, viral infection, and tumor biology. Thechemokines exert their effects by acting on a family of seventransmembrane G- protein coupled receptors. Over 40 human chemokineshave been described, which bind to ~17 receptors thus far identified.Chemokine activities can be determined using assays known in the art:Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols.Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. HumanaPress Inc., Totowa, NJ. Cancer and infectious diseases Human CKbeta-9Chemokines are a family of related small, secreted proteins involved inbiological GeneSeq Accession processes ranging from hematopoiesis,angiogenesis, and leukocyte trafficking. B50860 U.S. Pat. No. Members ofthis family are involved in a similarly diverse range of pathologies6,153,441 including inflammation, allergy, tissue rejection, viralinfection, and tumor biology. The O00585/CCL21_HUMAN chemokines exerttheir effects by acting on a family of seven transmembrane G- C-C motifchemokine 21 protein coupled receptors. Over 40 human chemokines havebeen described, which (SEQ ID NO: 346) bind to ~17 receptors thus faridentified. Chemokine activities can be determined using assays known inthe art: Methods in Molecular Biology, 2000, vol. 138: ChemokineProtocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A.Power. Humana Press Inc., Totowa, NJ. Cancer, Auto-immune andinflammatory disorders, Cardiovascular disorders Preproapolipo-proteinApoa-1 participates in the reverse transport of cholesterol from tissuesto the liver for “paris” variant GeneSeq excretion by promotingcholesterol efflux from tissues and by acting as a cofactor forAccession W08602 the lecithin cholesterol acyltransferase (lcat). Lipidbinding activity can be determined WO9637608 using assays known in theart, such as, for example, the Cholesterol Efflux Assays of (SEQ ID NO:466) Takahaski et al., P. N. A. S., Vol. 96, Issue 20, 11358-11363, Sep.28, 1999. Useful for cardio-vascular disorders, cholesterol disorders,and Hyperlipidaemia Preproapolipo-protein Apoa-1 participates in thereverse transport of cholesterol from tissues to the liver for “milano”variant 5,721,114 excretion by promoting cholesterol efflux from tissuesand by acting as a cofactor for SEQ ID NO: 6 of U.S. the lecithincholesterol acyltransferase (Icat). Lipid binding activity can bedetermined Pat. No. 5,721,114 using assays known in the art, such as,for example, the Cholesterol Efflux Assays of (SEQ ID NO: 441) Takahaskiet al., P. N. A. S., Vol. 96, Issue 20, 11358-11363, Sep. 28, 1999.Useful for cardio-vascular disorders, cholesterol disorders, andHyperlipidaemia Glycodelin-A; Naturally produced female contraceptivethat is removed rapidly from the body Progesterone-associated following2-3 days production. Uses include contraception Glycodelin-A activitycan endometrial protein be determined using the hemizona assay asdescribed in Oehninger, S., Coddington, C. C., GeneSeq Accession Hodgen,G. D., and Seppala, M (1995) Fertil. Steril. 63, 377-383. NaturallyW00289 WO9628169 derived contraceptive useful for the prevention ofpregnancy. P09466/PAEP_HUMAN Glycodelin (SEQ ID NO: 365) NOGO-A GenbankNOGO polypeptides are potent inhibitors of neurite growth. Inhibition ofNeurite Accession CAB99248 outgrowth. Antagonists to NOGO polypeptidesmay promote the outgrowth of (SEQ ID NO: 366) neurites, thus inducingregeneration of neurons. NOGO-A polypeptide antagonists are useful forthe promotion of neural growth, which could be useful in the treatmentof neural disorders and dys-function due to de-generative diseases ortrauma; useful in the treatment of neo-plastic diseases of the CNS;induce regeneration of neurons or to promote the structural plasticityof the CNS. NOGO-B Genbank NOGO polypeptides are potent inhibitors ofneurite growth. Inhibition of Neurite Accession CAB99249 outgrowth.Antagonists to NOGO polypeptides may promote the outgrowth of (SEQ IDNO: 367) neurites, thus inducing regeneration of neurons. NOGO-Bpolypeptide antagonists are useful for the promotion of neural growth,which could be useful in the treatment of neural disorders anddys-function due to de-generative diseases or trauma; useful in thetreatment of neo-plastic diseases of the CNS; induce regeneration ofneurons or to promote the structural plasticity of the CNS. NOGO-CGenbank NOGO polypeptides are potent inhibitors of neurite growth.Inhibition of Neurite Accession CAB99250 outgrowth. Antagonists to NOGOpolypeptides may promote the outgrowth of (SEQ ID NO: 368) neurites,thus inducing regeneration of neurons. NOGO-C polypeptide antagonistsare useful for the promotion of neural growth, which could be useful inthe treatment of neural disorders and dys-function due to de-generativediseases or trauma; useful in the treatment of neo-plastic diseases ofthe CNS; induce regeneration of neurons or to promote the structuralplasticity of the CNS. NOGO-66 Receptor NOGO polypeptides are potentinhibitors of neurite growth, and are thought to Genbank Accessionmediate their effects through the NOGO-66 Receptor. Inhibition ofNeurite outgrowth AAG53612 by mediating the biological effects of NOGOpolypeptides. Soluble NOGO-66 (SEQ ID NO: 369) receptor polypeptides maypromote the outgrowth of neurites, thus inducing regeneration ofneurons. NOGO-66 receptor polypeptides are useful for the promotion ofneural growth, which could be useful in the treatment of neuraldisorders and dys-function due to de-generative diseases or trauma;useful in the treatment of neo-plastic diseases of the CNS; induceregeneration of neurons or to promote the structural plasticity of theCNS. Antibodies specific for These antibodies are useful for thepromotion of neurite outgrowth Collapsin activity, collapsin U.S. Pat.No. which is thought to inhibit the outgrowth of neurites, can beassayed in the presence 5,416,197 of antibodies specific for collapsingusing assays known in the art, such as, for Wildtype collapsin has theexample, the collapse assay disclosed by Luo et al., Cell 1993 Oct. 22;75(2): 217-27 sequence: Useful for the promotion of neural growth, whichcould be useful in the treatment of SEQ ID NO: 2 of 5,416,197 neuraldisorders and dys-function due to de-generative diseases or trauma. (SEQID NO: 464) Humanized Anti-VEGF These agents have anti-inflammatory andanti-cancer applications VEGF activity can Antibodies, and fragments bedetermined using assays known in the art, such as those disclosed inInternational thereof WO9845331 Publication No. WO0045835, for example.Promotion of growth and proliferation of cells, such as vascularendothelial cells. Antagonists may be useful as anti- angiogenic agents,and may be applicable for cancer Humanized Anti-VEGF These agents haveanti-inflammatory and anti-cancer applications VEGF activity canAntibodies, and fragments be determined using assays known in the art,such as those disclosed in International thereof WO0029584 PublicationNo. WO0045835, for example. Promotion of growth and proliferation ofcells, such as vascular endothelial cells. Hematopoietic and immunedis-orders. Antagonists may be useful as anti-angiogenic agents, and maybe applicable for cancer Membrane bound proteins Cancer, ImmuneDisorders These proteins can be used for linking bioactive GeneSeq.Accession molecules to cells and for modulating biological activities ofcells, using the Y66631-Y66765 polypeptides for specific targeting. Thepolypeptide targeting can be used to kill the WO9963088 target cells,e.g. for the treatment of cancers. These proteins are useful for thetreatment of immune system disorders. Activities can be determined usingassay known in the art, such as, for example, the assays disclosed inInternational Publication No. WO0121658. Secreted and Cancer, ImmuneDisorders These proteins can be used for linking bioactive Transmembranemolecules to cells and for modulating biological activities of cells,using the polypeptides GeneSeq polypeptides for specific targeting. Thepolypeptide targeting can be used to kill the Accession B44241-B44334target cells, e.g. for the treatment of cancers. These proteins areuseful for the WO0053756 treatment of immune system disorders.Activities can be determined using assay known in the art, such as, forexample, the assays disclosed in International Publication No.WO0121658. Secreted and Cancer, Immune Disorders These proteins can beused for linking bioactive Transmembrane molecules to cells and formodulating biological activities of cells, using the polypeptidesGeneSeq polypeptides for specific targeting. The polypeptide targetingcan be used to kill the Accession Y41685-Y41774 target cells, e.g. forthe treatment of cancers. These proteins are useful for the WO9946281treatment of immune system disorders. Activities can be determined usingassay known in the art, such as, for example, the assays disclosed inInternational Publication No. WO0121658. Interleukin 2 (IL-2) MetabolicDisease, Type 1 diabetes, Graft-versus-host disease. SEQ ID NO: 548Interleukin 15_vA Obesity, Metabolic Disease, Diabetes. (IL-15_vA) SEQID NO: 549 Interleukin 15_vB Obesity, Metabolic Disease, Diabetes.(IL-15_vB) SEQ ID NO: 550 Interleukin 15_vC Obesity, Metabolic Disease,Diabetes. (IL-15_vC) SEQ ID NO: 551 Interleukin 15_vD Obesity, MetabolicDisease, Diabetes. (IL15_vD) SEQ ID NO: 552 Interleukin 15_vE Obesity,Metabolic Disease, Diabetes. (IL15_vE) SEQ ID NO: 553 Interleukin 15_vFObesity, Metabolic Disease, Diabetes. (IL15_vF) SEQ ID NO: 565Interleukin 22 Metabolic Disease, Diabetic Ulcers, Inflamatory BowelDiseases. (IL22) SEQ ID NO: 554 Fibroblast Growth Factor 1 Diabetes,Metabolic Disease, Obesity. (FGF1) SEQ ID NO: 555 Fibroblast GrowthFactor Diabetes, Metabolic Disease, Obesity. 1_vA See Nature 513,436-439 (18 Sep. 2014) doi: 10.1038/nature13540. (FGF1_vA) SEQ ID NO:556 Fibroblast Growth Factor Diabetes, Metabolic Disease, Obesity. 1_vBSee Proc Natl Acad Sci USA. 1991 Apr. 1; 88(7): 2893-2897. (FGF1_vB) SEQID NO: 557 Fibroblast Growth Factor Diabetes, Metabolic Disease,Obesity. 1_vC (FGF1_vC) SEQ ID NO: 566 Fibroblast Growth Factor Chronicliver disease, primary biliary cirrhosis, bile acid-induced liverdamage. 19_vA See Cancer Res. 2014 Jun. 15; 74(12): 3306-16. doi:10.1158/0008-5472.CAN-14- (FGF19_vA) 0208. Epub 2014 Apr. 11. Regulatesbile acid metabolism without tumorigenicity. SEQ ID NO: 558 FibroblastGrowth Factor Metabolic Disease, Fibrotic Diseases. 21 (FGF21) SEQ IDNO: 559 Fibroblast Growth Factor Hyperphosphatemic familial tumoralcalcinosis. 23 (FGF23) SEQ ID NO: 560 Brain-Derived Neurologicaldiseases (including Alzheimer's Disease, Autism, Huntington's Disease,Neurotrophic Factor Parkinson's Disease, and Depression), obesity,metabolic disease. (BDNF) SEQ ID NO: 561 Serpin Family A Member 1alpha-1-antitrypsin deficiency. (SERPINA1) SEQ ID NO: 584, SEQ ID NO:585 Serpin Peptidase Inhibitor, Diabetes. Clade B (Ovalbumin), Member 1(SERPINB1) SEQ ID NO: 562 CASPASE1 Diabetes. SEQ ID NO: 563 LeukemiaInhibitory Factor Muscular dystrophy, atherosclerosis, kidney disease(LIF) SEQ ID NO: 564 Proprotein Convertase Cardiovascular disease,hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 1hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease suchas heart (PCSK1) attacks or strokes, increasing the amount of afunctional protein, polypeptide or SEQ ID NO: 567 peptide ProproteinConvertase Cardiovascular disease, hypercholesterolemia, heterozygousfamilial Subtilisin/Kexin Type 2 hypercholesterolemia (HeFH),atherosclerotic cardiovascular disease such as heart (PCSK2) attacks orstrokes, increasing the amount of a functional protein, polypeptide orSEQ ID NO: 568 peptide Proprotein Convertase Cardiovascular disease,hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 3hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease suchas heart (PCSK3) attacks or strokes, increasing the amount of afunctional protein, polypeptide or SEQ ID NO: 569 peptide ProproteinConvertase Cardiovascular disease, hypercholesterolemia, heterozygousfamilial Subtilisin/Kexin Type 3 Sol hypercholesterolemia (HeFH),atherosclerotic cardiovascular disease such as heart (PCSK3_SOL) attacksor strokes, increasing the amount of a functional protein, polypeptideor SEQ ID NO: 570 peptide Proprotein Convertase Cardiovascular disease,hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 4hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease suchas heart (PCSK4) attacks or strokes, increasing the amount of afunctional protein, polypeptide or SEQ ID NO: 571 peptide ProproteinConvertase Cardiovascular disease, hypercholesterolemia, heterozygousfamilial Subtilisin/Kexin Type 5 hypercholesterolemia (HeFH),atherosclerotic cardiovascular disease such as heart (PCSK5) attacks orstrokes, increasing the amount of a functional protein, polypeptide orSEQ ID NO: 572 peptide Proprotein Convertase Cardiovascular disease,hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 6hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease suchas heart (PCSK6) attacks or strokes, increasing the amount of afunctional protein, polypeptide or SEQ ID NO: 573 peptide ProproteinConvertase Cardiovascular disease, hypercholesterolemia, heterozygousfamilial Subtilisin/Kexin Type hypercholesterolemia (HeFH),atherosclerotic cardiovascular disease such as heart (PCSK7) attacks orstrokes, increasing the amount of a functional protein, polypeptide orSEQ ID NO: 574 peptide Proprotein Convertase Cardiovascular disease,hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 8hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease suchas heart (PCSK8) attacks or strokes, increasing the amount of afunctional protein, polypeptide or SEQ ID NO: 575 peptide ProproteinConvertase Cardiovascular disease, hypercholesterolemia, heterozygousfamilial Subtilisin/Kexin Type 9 hypercholesterolemia (HeFH),atherosclerotic cardiovascular disease such as heart (PCSK9) attacks orstrokes, increasing the amount of a functional protein, polypeptide orSEQ ID NO: 576 peptide Membrane-Bound IFAP syndrome, increasing theamount of a functional protein, polypeptide or peptide TranscriptionFactor Peptidase, Site 2 (MBTPS2) SEQ ID NO: 577 Carboxypeptidase EEndocrine disorders, such as, for example, obesity and infertility;(CPE) hyperproinsulinemia; metabolic syndrome, increasing the amount ofa functional SEQ ID NO: 578 protein, polypeptide or peptide

In various embodiments, the nucleic acid drug, including synthetic RNA,is administered is a manner that it effects one or more of keratinocytesand fibroblasts (e.g. causes these cells to express one or moretherapeutic proteins).

For example, present methods allow for methods in which a patient'scells are used to generate a therapeutic protein and the levels of suchprotein are tailored by synthetic RNA dosing.

In a specific embodiment, the synthetic RNA targets a soluble protein.In some embodiments, the synthetic RNA targets a protein of one or moreof the following families of proteins: transforming growth factor (TGF)beta, bone morphogenetic proteins (BMPs), Fibroblast growth factors(FGFs), vascular endothelial growth factors (VEGFs), and interleukins.The terms “family”, “superfamily”, and “subfamily” can be usedinterchangeably

In a specific embodiment, the synthetic RNA targets a member of the TGFbeta family. TGF-β superfamily proteins comprise cytokines characterizedby six-conserved cysteine residues (Lander et al., (2001) Nature,409:860-921). The human genome contains at least about 42 open readingframes encoding TGF-β superfamily proteins. TGF-β superfamily proteinscan at least be divided into the BMP subfamily and the TGF-β subfamilybased on sequence similarity and the specific signaling pathways thatthey activate. In various embodiments, the synthetic RNA targets one ormore of TGFs (e.g., TGF-β1, TGF-β2, and TGF-β3), activins (e.g., activinA) and inhibins, macrophage inhibitory cytokine-1 (MIC-1), Mullerianinhibiting substance, anti-Mullerian hormone, and glial cell linederived neurotrophic factor (GDNF).

The TGF-β superfamily comprises a subset of the cysteine knot cytokinesuperfamily. Additional members of the cysteine knot cytokinesuperfamily include, but are not limited to, platelet derived growthfactor (PDGF), vascular endothelial growth factor (VEGF), placentagrowth factor (P1GF), Noggin, neurotrophins (BDNF, NT3, NT4, and βNGF),gonadotropin, follitropin, lutropin, interleukin-17, and coagulogen.This family of proteins is also among the targets encompassed by thepresent invention.

In various embodiments, the present invention relates to targeting a TGFbeta family member for treatment or prevention of various immunologicaldisorders, cancer, bronchial asthma, lung fibrosis, heart disease,diabetes, hereditary hemorrhagic telangiectasia, Marfan syndrome,Vascular Ehlers-Danlos syndrome, Loeys-Dietz syndrome, Parkinson'sdisease, chronic kidney disease, multiple sclerosis and AIDS.

In a specific embodiment, the synthetic RNA targets a member of the BMPfamily. The BMP subfamily includes, but is not limited to, BMP-2, BMP-3(osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b), BMP-5, BMP-6, BMP-7(osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B (OP-3), BMP-9(GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7), BMP-13 (GDF-6,CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1), and GDF-8(myostatin). In various embodiments, the synthetic RNA targets one ormore of BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b),BMP-5, BMP-6, BMP-7 (osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B(OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7), BMP-13(GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1),and GDF-8 (myostatin). BMPs are sometimes referred to as OsteogenicProtein (OPs), Growth Differentiation Factors (GDFs), orCartilage-Derived Morphogenetic Proteins (CDMPs). In a specificembodiment, the synthetic RNA targets one or more BMP fusions (e.g. asdescribed in US Patent Publication No. 2009/0202638, the entire contentsof which are hereby incorporated by reference) and/or one or more BMPmutants (e.g. as described in US Patent Publication No. 2011/0039773,the entire contents of which are hereby incorporated by reference).

In various embodiments, the present invention relates to targeting a BMPfamily member for regenerative medicine or metabolic applications,including without limitation orthopedic applications such as spinalfusions, nonunions and oral surgerie, metabolic disease, prediabetes,diabetes, thermogenesis, insulin sensitivity, insulin resistance, andadipogenesis, including brown-fat adipogenesis. Various other metabolicapplications are described elsewhere herein. In various embodiments, thepresent invention relates to targeting a BMP family member, includingBMP-7, for treatment of chronic kidney disease (CKD) and/or to reversethe loss of glomeruli due to sclerosis.

In various embodiments, the present invention relates to targeting a BMPfamily member to induce proliferation of bone and cartilage in a varietyof locations in the body. For example, repair of joints such as knee,elbow, ankle, and finger joints are contemplated by the invention. Forexample, targeting a BMP family member may result in regeneratingcartilage in patients suffering from arthritis or other cartilagedegenerating diseases. Further, the invention pertains to treating tearsin cartilage due to injury. In addition, the invention is useful forinducing bone growth in patients, for instance, by way ofnon-limitation, for use in treating patients suffering from bonefractures or breaks, osteoporosis, or patients in need of spinal fusionor for repair of the spine, vertebrae or the like.

In various embodiments, the present invention relates to targeting a BMPfamily member to induce a developmental cascade of bone morphogenesisand tissue morphogenesis for a variety of tissues in mammals differentfrom bone or bone cartilage. This morphogenic activity includes theability to induce proliferation and differentiation of progenitor cells,and the ability to support and maintain the differentiated phenotypethrough the progression of events that results in the formation of bone,cartilage, non-mineralized skeletal or connective tissues, and otheradult tissues.

For example, the present invention may be used for treatment to preventloss of and/or increase bone mass in metabolic bone diseases. Generalmethods for treatment to prevent loss of and/or increase bone mass inmetabolic bone diseases using osteogenic proteins are disclosed in U.S.Pat. No. 5,674,844, the entire contents of which are hereby incorporatedby reference. Further the present compositions and methods find use inreplacing or repairing bone or cartilage at injury sites such as bonebreaks, bone fractures, and cartilage tears, periodontal tissueregeneration (e.g. general methods for periodontal tissue regenerationusing osteogenic proteins are disclosed in U.S. Pat. No. 5,733,878, theentire contents of which are hereby incorporated by reference), liverregeneration, including following a partial hepatectomy (see, e.g., U.S.Pat. No. 5,849,686, the entire contents of which are hereby incorporatedby reference), treatment of chronic renal failure (see, e.g., U.S. Pat.No. 6,861,404, the entire contents of which are hereby incorporated byreference), enhancing functional recovery following central nervoussystem ischemia or trauma (see, e.g. U.S. Pat. No. 6,407,060, the entirecontents of which are hereby incorporated by reference), inducingdendritic growth (see, e.g., U.S. Pat. No. 6,949,505, the entirecontents of which are hereby incorporated by reference), inducing neuralcell adhesion (see, e.g., U.S. Pat. No. 6,800,603, the entire contentsof which are hereby incorporated by reference), and treatment andprevention of Parkinson's disease (see, e.g., U.S. Pat. No. 6,506,729,the entire contents of which are hereby incorporated by reference). Asanother example, the present compositions and methods, including whentargeting one or more BMPs, can be used to induce dentinogenesis. Todate, the unpredictable response of dental pulp tissue to injury is abasic clinical problem in dentistry. Using standard dental surgicalprocedures, small areas (e.g., 2 mm) of dental pulps can be surgicallyexposed by removing the enamel and dentin immediately above the pulp (bydrilling) of sample teeth, performing a partial amputation of thecoronal pulp tissue, inducing hemostasis, application of the pulptreatment, and sealing and filling the cavity by standard procedures.

In various embodiments, the present invention relates to targeting a BMPfamily member for the treatment of one or more metabolic-relateddisorders as described herein.

In a specific embodiment, the synthetic RNA targets a member of the FGFfamily. FGFs are a family of growth factors, with members involved inangiogenesis, wound healing, embryonic development and various endocrinesignaling pathways. In various embodiments, any one of the followingFGFs are targets of the invention: FGF1, FGF2, FGF3, FGF4, FGF5, FGF6,FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14 (FGF11, FGF12,FGF13, and FGF14 being FGF homologous factors 1-4 (FHF1-FHF4)), FGF16,FGF17, FGF18, FGF19 (aka FHF15/19), FGF20, FGF21, FGF22 and FGF22. Insome embodiments, the synthetic RNA targets a cognate receptor of any ofthe above FGFs. In various embodiments, the present invention relates totargeting a FGF family member to a treat or prevent disease or disorderassociated with abnormal function and/or expression of an FGF, ametabolic disease or disorder (e.g., diabetes, obesity, dyslipidemia,hyperglycemia, hyperinsulinemia, hypertension, hepatosteaotosis such asnon-alcoholic steatohepatitis (NASH) etc.), cancer, a disease ordisorder associated with impaired lipid metabolism a disease or disorderassociated with impaired renal function, a disease or disorderassociated with impaired hepatic function, abnormal cell proliferation,a vascular disease or disorder (e.g., coronary artery disease,peripheral artery disease, atherosclerosis, abdominal aortic aneurysm, ablood clot, deep vein thrombosis, venous stasis disease, phlebitis,varicose veins etc.), angiogenesis, atherosclerosis, a cardiovasculardisease or disorder, a disease or disorder associated with impairedblood vessel formation, a disease or disorder associated with impairedcell signaling, a disease or disorder associated with impaired kinaseactivity, and a disease or disorder associated with impaired uptake ofglucose into adipocytes.

In a specific embodiment, the synthetic RNA targets a member of the VEGFfamily. In some embodiments, the target is one or more of VEGF-A(including all isoforms, e.g. VEGF121, VEGF165 and VEGF189), placentagrowth factor (PGF, including all isoforms, e.g. PGF-1, PGF-2, andPGF-3), VEGF-B, VEGF-C and VEGF-D, and any variants thereof (see, e.g.U.S. Pat. No. 9,078,860, the entire contents of which are herebyincorporated by reference). In some embodiments, the synthetic RNAtargets a cognate receptor of any of the above VEGFs. The presentinvention also encompasses as targets VEGF-related proteins includingorf virus-encoded VEGF-like proteins referred to as VEGF-E and a seriesof snake venoms referred to as VEGF-F. VEGFs and VEGF-related proteinsare members of the Platelet Derived Growth Factor (PDGF) supergenefamily of cystine knot growth factors. All members of the PDGF supergenefamily share a high degree of structural homology with PDGF.

In various embodiments, the present invention relates to targeting aVEGF family member to treat diseases and conditions associated withangiogenesis, including but not limited to, solid tumor cancers,hemangiomas, rheumatoid arthritis, osteoarthritis, septic arthritis,asthma, atherosclerosis, idiopathic pulmonary fibrosis, vascularrestenosis, arteriovenous malformations, meningiomas, neovascularglaucoma, psoriasis, Kaposi's Syndrome, angiofibroma, hemophilic joints,hypertrophic scars, Osler-Weber syndrome, pyogenic granuloma,retrolental fibroplasias, scleroderma, trachoma, von Hippel-Lindaudisease, vascular adhesion pathologies, synovitis, dermatitis,neurological degenerative diseases, preeclampsia, unexplained femaleinfertility, endometriosis, unexplained male infertility, pterygium,wounds, sores, skin ulcers, gastric ulcers, and duodenal ulcers. Invarious embodiments, the present invention relates to targeting a VEGFfamily member to treat angiogenesis-associated eye diseases, includingwithout limitation any eye disease associated with abnormal intraocularneovascularization, including but not limited to retinopathy ofprematurity, diabetic retinopathy, retinal vein occlusion, andage-related macular degeneration, as well diabetic macular edema andretinal vein occlusion. In an embodiment, the present compositions andmethods relate to the treatment of wet age-related macular degeneration.

In a specific embodiment, the synthetic RNA targets a member of theinterleukin family. The interleukins represent a large group ofcytokines with diverse functions and were first characterized byexpression in leukocytes and have since been shown to be expressed in awide variety of cells, for example macrophages, TH-1 and TH-2 cells,T-lymphocytes, monocytes and bone marrow stroma. Broadly, the functionof the immune system depends in a large part on the expression andfunction of the interleukins. In some embodiments, the target is one ormore of interleukins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,35 and 36, and, within each species of interleukin, various isotypesand/or interleukin receptors (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R,IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R,IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R,IL-24R, IL-25R, IL-26, IL-27R, IL-28R, IL-29R, IL-30R, IL-31R, IL-32R,IL-33R, IL-34R, IL-35R, and IL-36R). In a specific embodiment, bothIL-15 and IL-15R (e.g., IL-15RA) are targeted. Without wishing to bebound by theory, it is believed that the targeting of both interleukin(e.g., IL-15) and its cognitive interleukin receptor (e.g., IL-15RA)results in synergistic beneficial effects. In various embodiments, thepresent invention relates to targeting a member of the interleukinfamily to treat cancer, inflammatory, respiratory, autoimmune,cardiovascular, neurological, metabolic, and/or proliferative diseases,disorders, and/or conditions in a subject or organism, as describedherein. In a specific embodiment, the present invention relates totargeting a member of the interleukin family to treat cancer. In aspecific embodiment, the present invention relates to targeting a memberof the interleukin family to treat rheumatoid arthritis.

In a specific embodiment, the synthetic RNA targets an EPO gene or aderivative thereof (e.g. SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166,and SEQ ID NO: 167). Some embodiments are related to NOVEPOETIN protein(SEQ ID NO: 167). Other embodiments are related to NOVECRIT (SEQ ID NO:168).

Erythropoietin can stimulate erythropoiesis in anemic patients withchronic renal failure in whom the endogenous production oferythropoietin is impaired. Without being bound by theory, because ofthe length of time often required for erythropoiesis (several days forerythroid progenitors to mature and be released into the circulation), aclinically significant increase in hemoglobin is usually not observed inless than two weeks and may require up to ten weeks in some patients.The present methods and compositions provide, in some embodiments, morerapid therapeutic effect. The present methods and compositions provide,in some embodiments, more rapid therapeutic effect, for instance, whencompared to wild type EPO and/or EPO without non-canonical nucleotidesand/or EPO delivered as a protein biologic. The present methods andcompositions provide, in some embodiments, a sustained therapeuticeffect. The present methods and compositions provide, in someembodiments, a sustained therapeutic effect, for instance, when comparedto wild type EPO and/or EPO without non-canonical nucleotides and/or EPOdelivered as a protein biologic.

For instance, for EPO, the present methods and compositions provide aclinically significant increase in hematocrit in less than about 6weeks, or less than about 5 weeks, or less than about 4 weeks, or lessthan about 3 weeks, or less than about 2 weeks, or less than about 1week. In some embodiments, the present methods and compositions providea clinically significant increase in hematocrit in about 2 weeks, orabout 10 days, or about 1 week, or about 3 days, or about 1 day. Invarious embodiments, the present methods and compositions accelerate theprocess by which erythroid progenitors mature and are released into thecirculation.

In some embodiments, the present EPO-related compositions find use indecreasing the dose and/or frequency of administration when compared towild type EPO and/or EPO without non-canonical nucleotides and/or EPOdelivered as a protein biologic. For instance, the present EPO-relatedcompositions may find use in treatment regimens for the diseasesdisclosed herein (including, without limitation, one or more anemias)that involve administration on a monthly, or biweekly, or weekly basis.In some embodiments, therefore, the present EPO-related compositionsreduce the need for daily, or, in some embodiments, weekly,administration. In some embodiments, the present EPO-relatedcompositions require lower maintenance doses as compared to wild typeEPO and/or EPO without non-canonical nucleotides and/or EPO delivered asa protein biologic. Certain embodiments are particularly useful fortreating chemotherapy-induced anemia. Other embodiments are particularlyuseful for treating anemia associated with inflammation, including, butnot limited to, rheumatoid arthritis.

In some embodiments, the present the present methods and compositionsprovide a fast and robust response that obviates the need for RBCtransfusion. For instance, in some embodiments, the present methods andcompositions allow for treatment of patients do not consent totransfusions.

In some embodiments, the present methods and compositions increase therate of increase in hematocrit. In some embodiments, the present methodsand compositions maintain elevated hematocrits (e.g. of 25%, or 30%, or35%, or 40% or more) for a sustained period (e.g. about 1 month, orabout 2 months, or about 3 months, or about 4 months, or about 5 months,or about 6 months, or about 9 months).

In some embodiments, the present methods and compositions stimulate redblood cell production. In some embodiments, the present methods andcompositions stimulate division and differentiation of committederythroid progenitors in the bone marrow.

In some embodiments, including, without limitation, when targeting EPO,the present invention relates to the treatment of one or more of anemia,including anemia resulting from resulting from chronic kidney disease(e.g. from dialysis) and/or chemotherapy and/or HIV treatment (e.g.Zidovudine (INN) or azidothymidine (AZT)), inflammatory bowel disease(e.g. Crohn's disease and ulcer colitis), anemia linked to inflammatoryconditions (e.g. arthritis, lupus, IBD), anemia linked to diabetes,schizophrenia, cerebral malaria, as aplastic anemia, and myelodysplasiafrom the treatment of cancer (e.g. chemotherapy and/or radiation), andvarious myelodysplastic syndrome diseases (e.g. sickle cell anemia,hemoglobin SC disease, hemoglobin C disease, alpha- andbeta-thalassemias, neonatal anemia after premature birth, and comparableconditions).

In some embodiments, including, without limitation, when targeting EPO,the present invention relates to the treatment of, or a patient havingone or more of cancer, heart failure, autoimmune disease, sickle celldisease, thalassemia, blood loss, transfusion reaction, diabetes,vitamin B12 deficiency, collagen vascular disease, Shwachman syndrome,thrombocytopenic purpura, Celiac disease, endocrine deficiency statesuch as hypothyroidism or Addison's disease, autoimmune disease such asCrohn's Disease, systemic lupus erythematosis, rheumatoid arthritis orjuvenile rheumatoid arthritis, ulcerative colitis immune disorders suchas eosinophilic fasciitis, hypoimmunoglobulinemia, or thymoma/thymiccarcinoma, graft vs. host disease, preleukemia, Nonhematologic syndrome(e.g. Down's, Dubowwitz, Seckel), Felty syndrome, hemolytic uremicsyndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria,osteomyelofibrosis, pancytopenia, pure red-cell aplasia,Schoenlein-Henoch purpura, malaria, protein starvation, menorrhagia,systemic sclerosis, liver cirrhosis, hypometabolic states, congestiveheart failure, chronic infections such as HIV/AIDS, tuberculosis,oseomyelitis, hepatitis B, hepatitis C, Epstein-barr virus orparvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungalor parasitic infections, and/or red cell membrane disorders such ashereditary spherocytosis, hereditary elliptocytosis, heriditraypyrpoikilocytosis, hereditary stomatocytosis, red cell enzyme defects,hypersplenism, immune hemolysis or paroxysmal nocturnal hemoglobinuria.

In some embodiments, including, without limitation, when targeting EPO,the present invention relates to the treatment of, or a patient havinganemia, i.e. a condition in which the number of red blood cells and/orthe amount of hemoglobin found in the red blood cells is below normal.In various embodiments, the anemia may be acute or chronic. For example,the present anemias include but are not limited to iron deficiencyanemia, renal anemia, anemia of chronic diseases/inflammation,pernicious anemia such as macrocytic achylic anemia, juvenile perniciousanemia and congenital pernicious anemia, cancer-related anemia,chemotherapy-related anemia, radiotherapy-related anemia, pure red cellaplasia, refractory anemia with excess of blasts, aplastic anemia,X-lined siderobalstic anemia, hemolytic anemia, sickle cell anemia,anemia caused by impaired production of ESA, myelodysplasia syndromes,hypochromic anemia, microcytic anemia, sideroblastic anemia, autoimmunehemolytic anemia, Cooley's anemia, Mediterranean anemia, DiamondBlackfan anemia, Fanconi's anemia and drug-induced immune hemolyticanemia. Anemia may cause serious symptoms, including hypoxia, chronicfatigue, lack of concentration, pale skin, low blood pressure, dizzinessand heart failure.

In some embodiments, the anemia is induced by chemotherapy is one causeof anemia. For instance, the chemotherapy may be any myelosuppressivechemotherapy. In some embodiments, the chemotherapy is one or moreplatinum-based drugs including cisplatin (e.g. PLATINOL) and carboplatin(e.g. PARAPLATIN). In some embodiments, the chemotherapy is any one ofthe agents described herein. In some embodiments, the chemotherapy isany agent described in Groopman et al. J Natl Cancer Inst (1999) 91(19): 1616-1634, the contents of which are hereby incorporated byreference in their entireties. In some embodiments, the presentcompositions and methods are used in the treatment ofchemotherapy-related anemia in later stage cancer patients (e.g. a stageIV, or stage III, or stage II cancer). In some embodiments, the presentcompositions and methods are used in the treatment ofchemotherapy-related anemia in cancer patients receiving dose-densechemotherapy or other aggressive chemotherapy regimens.

In some embodiments, the present invention relates to the treatment ofanemia in a patient having one or more blood-based cancers, such asleukemia, lymphoma, and multiple myeloma. Such cancers may affect thebone marrow directly. Further, the present invention relates tometastatic cancer that has spread to the bone or bone marrow. In someembodiments, the present invention relates to the treatment of anemia ina patient undergoing radiation therapy. Such radiation therapy maydamage the bone marrow, lowering its ability to make red blood cells. Infurther embodiments, the present invention relates to the treatment ofanemia in a patient having a reduction or deficiency of one or more ofiron, vitamin B12, and folic acid. In further embodiments, the presentinvention relates to the treatment of anemia in a patient havingexcessive bleeding including without limitation, after surgery or from atumor that is causing internal bleeding. In further embodiments, thepresent invention relates to the treatment of anemia in a patient havinganemia of chronic disease.

In some embodiments, the present invention relates to the treatment ofanemia resulting from chronic renal failure. In some embodiments, thepresent invention relates to the treatment of anemia resulting from theuse of one or more renal replacement therapies, inclusive of dialysis,hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration,and renal transplantation.

In some embodiments, the present invention relates to the treatment ofanemia in patients with chronic kidney disease who are not on dialysis.For instance, the present invention relates to patients in stage 1 CKD,or stage 2 CKD, or stage 3 CKD, or stage 4 CKD, or stage 5 CKD. In someembodiments, the present patient is stage 4 CKD or stage 5 CKD. In someembodiments, the present patient has undergone a kidney transplant. Insome embodiments, the present invention relates to the treatment ofanemia is a patient having an acute kidney injury (AKI).

In various embodiments, the present compositions and methods are used toreduce or eliminate fatigue, dizziness, and shortness of breath in apatient.

In various embodiments, the present compositions and methods are used totreat a patient presenting with hyporesponse or resistance toerythropoiesis stimulating agent therapy. In some embodiments,hyporesponsiveness to erythropoietin or ESA-resistant anemia refers tothe presence of at least one of the following conditions: i) asignificant decrease in hemoglobin levels at a constant dose of ESAtreatment, ii) a significant increase in the ESA dose requirement toachieve or maintain a certain hemoglobin level, iii) a failure to raisethe hemoglobin level to the target range despite the ESA dose equivalentto erythropoietin greater than 150 IU/kg/week or 0.75 mg/kg/week ofdarbepoeitn-alpha or continued need for such high dose of ESA tomaintain the target hemoglobin level. For example, approximately 5-10%of patients with CDK demonstrate hyporesponsiveness to ESA, defined as acontinued need for greater than 300 IU/kg per week erythropoietin or 1.5ng/kg per week darbepoetin administered by the subcutaneous route.

In various embodiments, the present compositions and methods mitigatethe need for dose-escalation of the erythropoiesis stimulating agenttherapy and therefore, optionally, avoid side effects (e.g. flu-likesymptoms such as joint pains, weakness, dizziness and tiredness, skinirritation, increased risk of adverse cardiovascular complications).

In various embodiments, the present compositions and methods are used tomaintain a hemoglobin level of about 12.5 to 13 g/dL. In variousembodiments, the present compositions and methods are used in patientshaving hemoglobin levels of below about 12 g/dL, or about 11 g/dL, orabout 10 g/dL, or about 9 g/dL, or about 8 g/dL, or about 7 g/dL, orabout 6 g/dL, or about 5 g/dL. In various embodiments, the presentcompositions and methods are used in patients having iron blood testscores that indicate blood pathology, e.g. a ferritin score of belowabout 200 ng/L and/or a transferrin saturation score below about 30%.

In various embodiments, the present compositions and methods are used toincrease or maintain hemoglobin levels at a target level ranging from 9to 10 g/dL, at a target level ranging from 9 g/dL to 11 g/dL, at atarget level ranging from 9 g/dL to 12 g/dL, at a target level rangingfrom 9 g/dL to 14 g/dL, at a target level ranging from 10 g/dL to 14g/dL, or at a target level ranging from 12 g/dL to 14 g/dL.

In various embodiments, the present compositions and methods are used tobring a patient's hemoglobin levels to normal. In various embodiments,normal hemoglobin ranges for humans are about 14-18 g/dl for men and12-16 for women g/dl with the average hemoglobin value for men at about16 g/dL and for women at about 14 g/dL.

In some embodiments, for instance when targeting EPO, the presentinvention relates to the treatment of anemia of one or more of thefollowing toxicity grading criteria (e.g. NCI Common Toxicity Criteria):grade 1 (mild), 10.0 g hemoglobin/dL to within normal limits; grade 2(moderate), 8.0-10.0 g of hemoglobin/dL; grade 3 (serious or severe),6.5-7.9 g of hemoglobin/dL; and grade 4 (life threatening), less than6.5 g of hemoglobin/dL. In various embodiments, the present inventionbrings an increase in toxicity grading criteria by about 1 point, orabout 2 points, or about 3 points, or about 4 points. In variousembodiments, the present invention results in a patient having a levelof 0 or 1. In various embodiments, the present compositions and methodsimprove anemia as assessed by one or more scales described in Groopmanet al. J Natl Cancer Inst (1999) 91 (19): 1616-1634, the entire contentsof which are hereby incorporated by reference in their entireties.

In some embodiments, when targeting EPO, the present invention relatesto combination therapy with one or more EPOs. For example, the presentcompositions may provide a sustained effect that can supplement a fastaction of another EPO. In some embodiments, the present compositions areused as an adjuvant to other EPOs. In some embodiments, the presentcompositions are used as a maintenance therapy to other EPOs Other EPOsinclude the following: epoetin alfa, including without limitation,DARBEPOETIN (ARANESP), EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGENPHARMACEUTICAL), EPOFIT (INTAS PHARMA), EPOGEN (AMGEN), EPOGIN, EPREX,(JANSSEN-CILAG), BINOCRIT (SANDOZ), PROCRIT; epoetin beta, includingwithout limitation, NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, Methoxypolyethylene glycol-epoetin beta (MIRCERA, ROCHE); epoetin delta,including without limitation, DYNEPO (erythropoiesis stimulatingprotein, SHIRE PLC); epoetin omega, including without limitation,EPOMAX; epoetin zeta, including without limitation, SILAPO (STADA) andRETACRIT (HOSPIRA) and other EPOs, including without limitation, EPOCEPT(LUPIN PHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE(BIOCON LTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR(EMCURE PHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTASBIOPHARMACEUTICA), WEPDX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFESCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCES), SHANPOIETIN (SHANTHABIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO)(SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

In some embodiments, when targeting EPO, the present invention relatesto combination therapy with a blood transfusion. For instance, thepresent compositions may supplement a blood transfusion. In someembodiments, when targeting EPO, the present invention relates tocombination therapy with iron supplements.

In some embodiments, the present invention also relates to the followingprotein targets, e.g. in the treatment of disease: growth hormone (GH)e.g. human and bovine growth hormone, growth hormone-releasing hormones;interferon including α-, β-, or γ-interferons, etc., interleukin-I;interleukin-II; erythropoietin including α- and β-erythropoietin (EPO),granulocyte colony stimulating factor (GCSF), granulocyte macrophagecolony stimulating factor (GM-CSF), anti-agiogenic proteins (e.g.,angiostatin, endostatin) PACAP polypeptide (pituitary adenylate cyclaseactivating polypeptide), vasoactive intestinal peptide (VIP),thyrotrophin releasing hormone (TRH), corticotropin releasing hormone(CRH), vasopressin, arginine vasopressin (AVP), angiotensin, calcitonin,atrial naturetic factor, somatostatin, adrenocorticotropin, gonadotropinreleasing hormone, oxytocin, insulin, somatotropin, plasminogen tissueactivator, coagulation factors including coagulation factors VIII andIX, glucosylceramidase, sargramostim, lenograstin, filgrastin,dornase-α, molgramostim, PEG-L-asparaginase, PEG-adenosine deaminase,hirudin, eptacog-α (human blood coagulation factor Vila) nerve growthfactors, transforming growth factor, epidermal growth factor, basicfibroblast growth factor, VEGF; heparin including low molecular weightheparin, calcitonin; antigens; monoclonal antibodies; vancomycin;desferrioxamine (DFO); parathyroid hormone, an immunogen or antigen, andan antibody such as a monoclonal antibody.

In some embodiments, the present methods allow for effective additionaltherapeutic agent (e.g. those described herein) activity and/ortargeting to a cell and/or tissue of interest. For example, the presentsynthetic RNA can lead to increased expression of one or more targetingmolecules that direct an additional therapeutic to the location oftherapy. For example, the additional therapeutic agent may have abinding partner that the synthetic RNA encodes. For example, thesynthetic RNA may induce the expression of an antigen that directs thetherapeutic activity of an antibody that may be used in combination(e.g. herceptin, rituxan, campath, gemtuzumab, herceptin, panorex,rituximab, bexxar, edrecolomab, alemtuzumab, mylotrag, IMC-C225, smartin195, and mitomomab). In some embodiments, the synthetic RNA can beinjected directly into one or more of the tumors described herein andhome the therapeutic antibody to the tumor.

In some embodiments, the present methods allow for effective additionaltherapeutic agent generation, especially when the additional therapeuticagent is a prodrug, for example, to produce an active form of the drug.In some embodiments, the synthetic RNA can be injected directly into oneor more of the tumors described herein and home the prodrug to thetumor. For instance, the synthetic RNA may encode an enzyme thatcatalyzes the localized conversion of a non-toxic, systemicallydelivered agent into a potent chemotherapeutic agent. By way ofillustration (note that any of the prodrugs or drugs listed herein areadditional agents as used herein):

Enzyme Prodrug Drug aldehyde oxidases 5-Ethynyl-2(1H)-pyrimidinone5-Ethynyluracil aldehyde oxidases IPdR IUdR aldehyde oxidases 5-FP 5-FUamino acid oxidases d-alanine Hydrogen peroxide amino acid oxidasesSeCys conjugates Selenols and hydrogen peroxide cytochrome P450reductase Menadione Semiquinone radical cytochrome P450 reductaseMitomycin C Quinone methide intermediate cytochrome P450 reductaseTirapazamine Nitroxide radical cytochrome P450 reductase EO9Unidentified semiquinone radical DT-diaphorase StreptonigrinUnidentified DT-diaphorase Mitomycin C Quinone methide intermediateDT-diaphorase CB 1954 5-(Aziridin-1-yl)-4-hydroxyl-amino-2-nitrobenzamide DT-diaphorase Diaziquone Semiguinone radical^(a)cytochrome P450 Ipomeanol Unidentified (possibly furan epoxide)cytochrome P450 Ftorafur (tegafur) 5-FU cytochrome P450 DacarbazineHHMTIC cytochrome P450 Trofosfamide Trofosfamide mustard cytochrome P450Ifosfamide Isophosphamide mustard cytochrome P450 CyclophosphamidePhosphoramide mustard cytochrome P450 AQ4N AQ4 tyrosinase2,4-Dihydroxyphenylalanine 6-Hydroxydopa tyrosinase 4-S-CAP BQtyrosinase GHB GBQ tyrosinase Substituted phenols Orthoquinonestyrosinase Phenyl mustards Phenol mustard tyrosinase Urea mustardsUnidentified glutathione S-transferase TER286 Aziridinium agentglutathione S-transferase S-CPHC-ethylsulfoxide S-CPHC-glutathioneglutathione S-transferase PTA 6-MP carboxylesterase CPT-11 SN-38carboxylesterase Paclitaxel-2-ethylcarbonate Paclitaxel carboxylesteraseCapecitabine 5′-Deoxy-5-fluorocytidine (5-FU) carboxylesterase Tertiaryamidomethyl esters Carboxylic acids and amines^(a) alkaline phosphataseAmifostine WR-1065 alkaline phosphatase 3-AP phosphate 3-APβ-glucuronidase BHAMG pHAM β-glucuronidase Epirubicin-glucuronideEpirubicin β-glucuronidase HMR 1826 Doxorubicin β-glucuronidase DNR-GA3Daunorubicin β-glucuronidase DOX-GA3 Doxorubicin β-glucuronidasePaclitaxel glucuronide Paclitaxel β-glucuronidase 5-FU glucuronide 5-FUcysteine conjugate β-lyase PC 6-MP cysteine conjugate β-lyase GC6-Thioguanine cysteine conjugate β-lyase SeCys conjugate SelenolNitroreductase CB 1954 5-(Aziridin-1-yl)-4- hydroxyl-amino-2- nitro-benzamide Cytochrome P450 4-Ipomeanol Unidentified (furan epoxide isspeculated) Cytochrome P450 Ifosfamide Isophosphoramide mustardCytochrome P450 Cyclophosphamide Phosphoramide mustard Purine-nucleosideFludarabine 2-Fluoroadenine phosphorylase Purine-nucleoside MeP-dR MePphosphorylase Thymidine kinase Ganciclovir Ganciclovir-triphosphatenucleotide Alkaline phosphatase Etoposide phosphate Etoposide Alkalinephosphatase Mitomycin C phosphate Mitomycin C Alkaline phosphatase POMPPOM Alkaline phosphatase N-(4-phosphonooxy)- Doxorubicinphenylacetyl)doxorubicin β-Glucuronidase Glucuronidated Nornitrogenmustard Oxazolidinone β-Glucuronidase Glucuronidated 9-amino-9-Aminocamptothecin camptothecin β-Glucuronidase Glucuronide mustardMustard Carboxypeptidase Methotrexate-amino acids MethotrexateCarboxypeptidase CMDA Benzoic acid mustard Penicillin amidase DPODoxorubicin Penicillin amidase MelPO Melphalan Penicillin amidase NHPAPPalytoxin Penicillin amidase N-(phenylacetyl) doxorubicin DoxorubicinPenicillin amidase N-(phenylacetyl) melphalan Melphalan β-Lactamase LY266070 DAVLBHYD β-Lactamase C-DOX Doxorubicin β-Lactamase PRODOXDoxorubicin β-Lactamase CM Phenylenediamine mustard β-Lactamase CCMPhenylenediamine mustard β-Lactamase Cephalosporin-DACCP DACCPβ-Lactamase PROTAX Taxol β-Lactamase Cephalosporin mitomycin C MitomycinC β-Lactamase C-Mel Melphalan Cytosine deaminase 5-Fluorocytosine5-Fluorouracil Methionine γ-lyase Selenomethionine MethylselenolMethionine γ-lyase Trifluoromethionine CSF₂

In certain embodiments, the synthetic RNA may encode an enzyme thatcatalyzes the conversion of 5-FU and/or Doxorubicin from variousprodrugs, as illustrated by the examples below:

5-FU Prodrugs and Enzymes 5-FP Aldehyde oxidase Ftorafur P450 5′-DFURThymidine phosphorylase 5-FU glucuronide β-Glucuronidase 5-FC Cytosinedeaminase Doxorubicin Prodrugs and EnzymesN-(4-phosphono-oxy)-phenylacetyl) Alkaline phosphatase doxorubicin HMR1826 β-Glucuronidase DOX-GA3 β-Glucuronidase DPO Penicillin amidaseN-(phenyacetyl) doxorubicin Penicillin amidase C-DOX β-Lactamase PRODOXβ-Lactamase

In some embodiments, the nucleic acid drugs at the doses and regimensdescribed herein may be used in combination with one or more additionalagents (aka adjuvant therapy or combination agent). In some embodiments,the nucleic acid drugs at the doses and regimens described herein may beused in a human patient undergoing treatment with one or more additionalagents. In some embodiments, the nucleic acid drug is used as anadjuvant or neoadjuvant to any of the additional agents describedherein. In some embodiments, the invention pertains to co-administrationand/or co-formulation. Any of the compositions described herein may beco-formulated and/or co-administered. In some embodiments, any nucleicacid drug described herein acts synergistically when co-administeredwith another agent and may be administered at doses that are lower thanthe doses commonly employed when such agents are used as monotherapy.

In some embodiments, any nucleic acid drug described herein may includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the composition such that covalent attachment doesnot prevent the activity of the composition. For example, but not by wayof limitation, derivatives include composition that have been modifiedby, inter alia, glycosylation, lipidation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of turicamycin,etc. Additionally, the derivative can contain one or more non-classicalamino acids. In various embodiments, one or more additional agents (akaadjuvant therapy or combination agent) may be conjugated to any nucleicacid drug described herein.

Contacting a cell with a steroid can suppress the innate immune responseto foreign nucleic acids, and can increase the efficiency of nucleicacid delivery and translation. Certain embodiments are thereforedirected to contacting a cell with a steroid. Other embodiments aredirected to administering a steroid to a patient. Illustrative steroidsinclude corticosteroid steroids. In some embodiments, the steroid is oneor more of cortisone, hydrocortisone, prednisone, prednisolone,dexamethasone, triamcinolone, and betamethasone. In one embodiment, thesteroid is hydrocortisone. In another embodiment, the steroid isdexamethasone.

Other embodiments are directed to administering to a patient a member ofthe group: an antibiotic, an antimycotic, and an RNAse inhibitor.

Botulinum toxin type A has been approved by the US Food and DrugAdministration (FDA) for the treatment of essential blepharospasm,strabismus and hemifacial spasm in patients over the age of twelve,cervical dystonia, glabellar line (facial) wrinkles and for treatinghyperhydrosis and botulinum toxin type B has been approved for thetreatment of cervical dystonia. The present compositions may be combinedwith these toxins in the treatment of these diseases and relateddiseases. Some embodiments are directed to a nucleic acid drug targetinga neurotoxin. In various embodiments, the neurotoxin is a botulinumtoxin or a biologically active fragment, variant, analogue orfamily-member thereof.

Further the combination of any one of the aforementioned toxins may beused in combination with the present compositions for various cosmeticprocedures, including, without limitation, facial wrinkles, hyperkineticskin lines, glabellar lines, crow's feet, marionette lines, skindisorders, nasolabial folds, blepharospasm, strabismus, hemifacialspasms and sweating disorders. Alternatively, the present compositionsmay be used to in these cosmetic procedures as a monotherapy.

Certain embodiments are directed to a combination therapy comprising oneor more of the therapeutic or cosmetic compositions of the presentinvention and one or more adjuvant therapies or cosmetic treatments. Incertain embodiments, one or more of the therapeutic or cosmeticcompositions of the present invention are administered to a subjectwhich is undergoing treatment with one or more adjuvant therapies orcosmetic treatments. Example adjuvant therapies and cosmetic treatmentsare set forth in Table 3 and Table 5 of U.S. Provisional Application No.61/721,302, the contents of which are hereby incorporated by reference,and are given by way of example, and not by way of limitation.

TABLE 3 Illustrative Adjuvant Therapies Example Therapy/Treatment ClassDisease/Condition Therapy/Treatment Acetylcholinesterase inhibitorsMyasthenia gravis, Glaucoma, Edrophonium Alzheimer′s disease, Lewy bodydementia, Postural tachycardia syndrome Angiotensin-converting-enzymeHypertension, Congestive heart failure Perindopril inhibitor Alkylatingagents Cancer Cisplatin Angiogenesis inhibitors Cancer, Maculardegeneration Bevacizumab Angiotensin II receptor antagonistsHypertension, Diabetic nephropathy, Valsartan Congestive heart failureAntibiotics Bacterial infection Amoxicillin Antidiabetic drugs DiabetesMetformin Antimetabolites Cancer, Infection 5-fluorouracil (5FU)Antisense oligonucleotides Cancer, Diabetes, Amyotrophic lateralMipomersen sclerosis (ALS), Hypercholesterolemia Cytotoxic antibioticsCancer Doxorubicin Deep-brain stimulation Chronic pain, Parkinson′sdisease, N/A Tremor, Dystonia Dopamine agonists Parkinson′s disease,Type II diabetes, Bromocriptine Pituitary tumors Entry/Fusion inhibitorsHIV/AIDS Maraviroc Glucagon-like peptide-1 agonists Diabetes ExenatideGlucocorticoids Asthma, Adrenal insufficiency, DexamethasoneInflammatory diseases, Immune diseases, Bacterial meningitisImmunosuppressive drugs Organ transplantation, Inflammatory Azathioprinediseases, Immune diseases Insulin/Insulin analogs Diabetes NPH insulinIntegrase inhibitors HIV/AIDS Raltegravir MAO-B inhibitors Parkinson′sdisease, Depression, Selegiline Dementia Maturation inhibitors HIV/AIDSBevirimat Nucleoside analog reverse- HIV/AIDS, Hepatitis B Lamivudinetranscriptase inhibitors Nucleotide analog reverse- HIV/AIDS, HepatitisB Tenofovir transcriptase inhibitors Non-nucleosidereverse-transcriptase HIV/AIDS Rilpivirine inhibitors Pegylatedinterferon Hepatitis B/C, Multiple sclerosis Interferon beta-1a Plantalkaloids/terpenoids Cancer Paclitaxel Protease inhibitors HIV/AIDS,Hepatitis C, Other viral Telaprevir infections Radiotherapy CancerBrachytherapy Renin inhibitors Hypertension Aliskiren StatinsHypercholesterolemia Atorvastatin Topoisomerase inhibitors CancerTopotecan Vasopressin receptor antagonist Hyponatremia, Kidney diseaseTolvaptan Dermal filler Wrinkles, aged skin Hyaluronic Acid Botulinumtoxin Wrinkles, aged skin botulinum toxin type A Induction of skinrepair Acne scars, aged skin Laser treatment, dermabrasion

In some embodiments, the additional agent is a cytotoxic agent,comprising, in illustrative embodiments, a toxin, a chemotherapeuticagent, a radioisotope, and an agent that causes apoptosis or cell death.

Illustrative cytotoxic agents include, but are not limited to,methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine; alkylating agents such as mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C,lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide,mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin(paraplatin); anthracyclines include daunorubicin (formerly daunomycin),doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin,epirubicin, mitoxantrone and bisantrene; antibiotics includedactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, andanthramycin (AMC); and antimytotic agents such as the vinca alkaloids,vincristine and vinblastine. Other cytotoxic agents include paclitaxel(taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B,gramicidin D, ethidium bromide, emetine, etoposide, tenoposide,colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxicagents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGFantagonists, EGFR antagonists, platins, taxols, irinotecan,5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide,melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesineand vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy,sex hormone antagonists, selective androgen receptor modulators,selective estrogen receptor modulators, PDGF antagonists, TNFantagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12Rantagonists, Toxin conjugated monoclonal antibodies, tumor antigenspecific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN, or anycombination thereof. Toxic enzymes from plants and bacteria such asricin, diphtheria toxin and Pseudomonas toxin may be conjugated to thetherapeutic agents (e.g. antibodies) to generatecell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad.Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419(1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described byGoldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention alsorelate to radioimmunoconjugates where a radionuclide that emits alpha orbeta particles is stably coupled to the antibody, or binding fragmentsthereof, with or without the use of a complex-forming agent. Suchradionuclides include beta-emitters such as Phosphorus-32, Scandium-47,Copper-67, Gallium-67, Yttrium-88, Yttrium-90, Iodine-125, Iodine-131,Samarium-153, Lutetium-177, Rhenium-186 or Rhenium-188, andalpha-emitters such as Astatine-211, Lead-212, Bismuth-212, Bismuth-213or Actinium-225.

Illustrative detectable moieties further include, but are not limitedto, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase,beta-galactosidase and luciferase. Further exemplary fluorescentmaterials include, but are not limited to, rhodamine, fluorescein,fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine,phycoerythrin and dansyl chloride. Further exemplary chemiluminescentmoieties include, but are not limited to, luminol. Further exemplarybioluminescent materials include, but are not limited to, luciferin andaequorin. Further exemplary radioactive materials include, but are notlimited to, Iodine-125, Carbon-14, Sulfur-35, Tritium and Phosphorus-32.

The dosage of any additional agent described herein as well as thedosing schedule can depend on various parameters, including, but notlimited to, the human patient's general health, and the administeringphysician's and/or human patient's discretion. Co-administration may besimultaneous or sequential. Any additional agent described herein, canbe administered prior to (e.g., about 5 minutes, about 15 minutes, about30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours,about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3weeks, about 4 weeks, about 5 weeks, about 6 weeks, 8 weeks, or about 12weeks before), concurrently with, or subsequent to (e.g., about 5minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6weeks, about 8 weeks, or about 12 weeks after) the administration of thenucleic acid drug to a human patient in need thereof. In variousembodiments any agent described herein is administered about 1 minuteapart, about 10 minutes apart, about 30 minutes apart, less than about 1hour apart, about 1 hour apart, about 1 hour to about 2 hours apart,about 2 hours to about 3 hours apart, about 3 hours to about 4 hoursapart, about 4 hours to about 5 hours apart, about 5 hours to about 6hours apart, about 6 hours to about 7 hours apart, about 7 hours toabout 8 hours apart, about 8 hours to about 9 hours apart, about 9 hoursto about 10 hours apart, about 10 hours to about 11 hours apart, about11 hours to about 12 hours apart, no more than about 24 hours apart orno more than about 48 hours apart.

In a specific embodiment, the combination regimen is designed to exploitthe finding that the nucleic acid drug dosing and formulation of thepresent invention has potent effects quickly (e.g. in about 6, or about12, or about 24, or about 30, or about 36, or about 48 hours) and theeffects can last about 7 days, or longer.

The dose of a nucleic acid drug is disclosed herein. In general, thedose of any additional agent that is useful is known to those in theart. For example, doses may be determined with reference Physicians'Desk Reference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011),the contents of which are incorporated by reference in its entirety. Insome embodiments, the present invention allows a patient to receivedoses that exceed those determined with reference Physicians' DeskReference. The dosage of any additional agent described herein candepend on several factors including the severity of the condition,whether the condition is to be treated or prevented, and the age,weight, and health of the human patient to be treated. Additionally,pharmacogenomic (the effect of genotype on the pharmacokinetic,pharmacodynamic or efficacy profile of a therapeutic) information abouta particular human patient may affect dosage used. Furthermore, theexact individual dosages can be adjusted somewhat depending on a varietyof factors, including the specific combination of the agents beingadministered, the time of administration, the route of administration,the nature of the formulation, the rate of excretion, the particulardisease being treated, the severity of the disorder, and the anatomicallocation of the disorder. Some variations in the dosage can be expected.

Cells, tissues, organs, and organisms, including, but not limited to,humans, have several characteristics that can inhibit or prevent thedelivery of nucleic acids, including, for example, the stratum corneum,which can serve as a barrier to foreign organisms and nucleic acids.These characteristics can thus inhibit the effects of therapeutics andcosmetics comprising nucleic acids. It has now been discovered that manyof these characteristics can be circumvented or overcome using a patchcomprising a flexible membrane and a plurality of needles, and that sucha patch can serve as an effective and safe article for the delivery ofnucleic acids. Certain embodiments are therefore directed to a nucleicacid delivery patch. In one embodiment, the nucleic acid delivery patchcomprises a flexible membrane. In another embodiment, the nucleic aciddelivery patch comprises a plurality of needles. In yet anotherembodiment, the plurality of needles are attached to the flexiblemembrane. In some embodiments, the patch comprises a nucleic acid. Inone embodiment, the nucleic acid is present in solution. In oneembodiment, the plurality of needles include one or more needles havinga lumen. In another embodiment, the patch further comprises a secondflexible membrane. In yet another embodiment, the flexible membrane andthe second flexible membrane are arranged to form a cavity. In a furtherembodiment, the cavity contains a nucleic acid. In a still furtherembodiment, the membrane comprises one or more holes through which anucleic acid can pass. In a still further embodiment, one or more holesand one or more needles having a lumen are arranged to allow the passageof a solution containing a nucleic acid through at least one of the oneor more holes and through at least one of the one or more needles havinga lumen. In some embodiments, the patch is configured to deliver asolution to the skin. In one embodiment, the solution comprises anucleic acid. In another embodiment, the solution comprises a vehicle.In yet another embodiment, the vehicle is a lipid or lipidoid. In astill further embodiment, the vehicle is a lipid-based transfectionreagent.

The cell membrane can serve as a barrier to foreign nucleic acids. Ithas now been discovered that combining the patch of the presentinvention with an electric field can increase the efficiency of nucleicacid delivery. Certain embodiments are therefore directed to a nucleicacid delivery patch comprising a plurality of needles, wherein at leasttwo needles form part of a high-voltage circuit. Certain embodiments aredirected to an implantable “tattoo” for microneedle delivery (see, e.g.Nature Materials 12, pp 367-376 (2013), the contents of which are herebyincorporated by reference in their entirety). In one embodiment, thehigh-voltage circuit generates a voltage greater than about 10V. Inanother embodiment, the high-voltage circuit generates a voltage greaterthan about 20V. In yet another embodiment, an electric field is producedbetween two of the needles. In a further embodiment, the magnitude ofthe electric field is at least about 100V/cm. In a still furtherembodiment, the magnitude of the electric field is at least about200V/cm. In some embodiments, the patch is configured to deliver anucleic acid to the epidermis. In other embodiments, the patch isconfigured to deliver a nucleic acid to the dermis. In still otherembodiments, the patch is configured to deliver a nucleic acid tosub-dermal tissue. In still other embodiments, the patch is configuredto deliver a nucleic acid to muscle. Certain embodiments are directed toa nucleic acid delivery patch comprising a plurality of electrodes. Inone embodiment, the plurality of electrodes is attached to a flexiblemembrane. Other embodiments are directed to a nucleic acid deliverypatch comprising a rigid structure. In one embodiment, a plurality ofelectrodes are attached to the rigid structure.

In some embodiments, the compositions described herein are administeredusing an array of needles covering an affected area of the subject. Insome embodiments, the treatment area is mechanically massaged afteradministration. In some embodiments, the treatment area is exposed toelectric pulses after administration. In some embodiments, the electricpulses are between about 10V and about 200V for from about 50microseconds to about 1 second. In some embodiments, the electric pulsesare generated around the treatment area by a multielectrode array.

In some embodiments, the present invention provides a patch deliverysystem, comprising a non-viral RNA transfection composition enclosedwithin a membrane, and an array of delivery needles delivering fromabout 10 ng to about 2000 ng of RNA per treatment area of about 100 cm²or less, or about 50 cm² or less, or about 10 cm² or less, or about 5cm² or less, or about 1 cm² or less, or about 0.5 cm² or less, or about0.2 cm² or less. In some embodiments, the non-viral transfectioncomposition contains from about 10 ng to about 2000 ng per injectionvolume of about 20 μL to about 1 ml. In some embodiments, each needledelivers an injection volume of between 1 μL and 500 μL.

In some embodiments, the delivery patch comprises an acrylic reservoirthat holds the nucleic acid drug. In some embodiments, a siliconadhesive is added to create a semisolid suspension of microscopic,concentrated drug cells. Further, some embodiments pride a patch that isassociated with one or more enhancers (these include, withoutlimitation, iontophoresis, ultrasound, chemicals including gels,microneedles, sonophoresis, lasers, and electroporatic methods).

In some embodiments, the delivery is effected via a gel, optionally ahydro alcoholic gel containing a combination of enhancers (e.g. COMBIGEL(ANTARES PHARMA)).

In various embodiments, the RNA is delivered using needle arrays.Illustrative needle arrays include, but are not limited to AdminPen 600and those described in U.S. Pat. Nos. 7,658,728, 7,785,301, and8,414,548, the entire disclosure of which are hereby incorporated byreference. Other examples of needles include, for example, the 3M™Hollow Microstructured Transdermal System and the 3M SolidMicrostructured Transdermal Systems (sMTS). See, e.g. U.S. Pat. Nos.3,034,507 and 3,675,766; Microneedles for Transdermal Drug Delivery.Advanced Drug Delivery Reviews. 56: 581-587 (2004); Pharm Res. 2011January; 28(1): 31-40, the entire contents of which are herebyincorporated by reference in their entireties.

In some embodiments, microneedles and/or microneedle arrays may be used.In various embodiments, the microneedles and/or microneedle arrays maybe, without limitation, solid, RNA-coated, dissolving, biodegradable,and/or hollow. In some embodiments, the delivery is effected via amicroneedle system, optionally combined with an electronicallycontrolled micropump that delivers the drug at specific times or upondemand. For example, the MACROFLUX (Alza) system may be used.

Other embodiments are directed to a method for delivering a nucleic acidto a cell in vivo comprising applying a nucleic acid to a tissuecontaining a cell in vivo. In one embodiment, the method furthercomprises applying a transient electric field in the vicinity of thecell. In another embodiment, the method results in the cell in vivointernalizing the nucleic acid. In yet another embodiment, the nucleicacid comprises synthetic RNA. In a further embodiment, the methodfurther results in the cell internalizing a therapeutically orcosmetically effective amount of the nucleic acid. In one embodiment,the cell is a skin cell. In another embodiment, the cell is a musclecell. In yet another embodiment, the cell is a dermal fibroblast. In afurther embodiment, the cell is a keratinocyte. In a still furtherembodiment, the cell is a myoblast. In some embodiments, the nucleicacid comprises a protein of interest. In one embodiment, the protein ofinterest is a fluorescent protein. In another embodiment, the protein ofinterest is an extracellular-matrix protein. In yet another embodiment,the protein of interest is a member of the group: elastin, collagen,laminin, fibronectin, vitronectin, lysyl oxidase, elastin bindingprotein, a growth factor, fibroblast growth factor, transforming growthfactor beta, granulocyte colony-stimulating factor, a matrixmetalloproteinase, an actin, fibrillin, microfibril-associatedglycoprotein, a lysyl-oxidase-like protein, platelet-derived growthfactor, a lipase, an uncoupling protein, thermogenin, filaggrin, afibroblast growth factor, an antibody, and a protein involved withpigment production. In some embodiments, the method further comprisesdelivering the nucleic acid to the epidermis. In other embodiments, themethod further comprises delivering the nucleic acid to the dermis. Instill other embodiments, the method further comprises delivering thenucleic acid below the dermis. In one embodiment, the delivering is byinjection. In another embodiment, the delivering is by injection using amicroneedle array. In yet another embodiment, the delivering is bytopical administration. In a further embodiment, the deliveringcomprises disruption or removal of a part of the tissue. In a stillfurther embodiment, the delivering comprises disruption or removal ofthe stratum corneum. In some embodiments, the nucleic acid is present insolution. In one embodiment, the solution comprises a growth factor. Inanother embodiment, the growth factor is a member of the group: afibroblast growth factor and a transforming growth factor. In yetanother embodiment, the growth factor is a member of the group: basisfibroblast growth factor and transforming growth factor beta. In otherembodiments, the solution comprises cholesterol.

In another embodiment, the method further comprises contacting the cellwith one or more nucleic acid molecules. In yet another embodiment, atleast one of the one or more nucleic acid molecules encodes a protein ofinterest. In a further embodiment, the method results in the cellexpressing the protein of interest. In a still further embodiment, themethod results in the cell expressing a therapeutically or cosmeticallyeffective amount of the protein of interest.

In another embodiment, the cell is contacted with a nucleic acidmolecule. In yet another embodiment, the method results in the cellinternalizing the nucleic acid molecule. In a further embodiment, themethod results in the cell internalizing a therapeutically orcosmetically effective amount of the nucleic acid molecule. In oneembodiment, the nucleic acid encodes a protein of interest. In oneembodiment, the nucleic acid molecule comprises a member of the group: adsDNA molecule, a ssDNA molecule, a RNA molecule, a dsRNA molecule, assRNA molecule, a plasmid, an oligonucleotide, a synthetic RNA molecule,a miRNA molecule, an mRNA molecule, and an siRNA molecule. In variousembodiments, the RNA comprises one or more non-canonical nucleotides.

In some embodiments, the present invention relates to one or moreadministration techniques described in U.S. Pat. Nos. 5,711,964;5,891,468; 6,316,260; 6,413,544; 6,770,291; and 7,390,780, the entirecontents of which are hereby incorporated by reference in theirentireties.

The invention also provides kits that can simplify the administration ofthe nucleic acid drugs described herein and/or any additional agentdescribed herein. An illustrative kit of the invention comprises anucleic acid drug and/or any additional agent described herein in unitdosage form. In one embodiment, the unit dosage form is a container,such as a pre-filled syringe, which can be sterile, containing any agentdescribed herein and a pharmaceutically acceptable carrier, diluent,excipient, or vehicle. The kit can further comprise a label or printedinstructions instructing the use of any agent described herein. The kitor one or more components of the kit may be stored at room temperature,about 4° C., about −20° C., about −80° C., or about −196° C. The kit mayalso include a lid speculum, topical anesthetic, and a cleaning agentfor the administration location. The kit can also further comprise oneor more additional agent described herein. In one embodiment, the kitcomprises a container containing an effective amount of a nucleic aciddrug as disclosed herein and an effective amount of another composition,such as an additional agent as described herein. In some embodiments,the unit dosage form is a pre-loaded (a.k.a. pre-dosed or pre-filled)syringe or a pen needle injector (injection pen)). Such unit dosageforms may comprise the effective doses of nucleic acid drug describedherein, e.g. about 10 ng to about 2000 ng, e.g. about 10 ng, or about 20ng, or about 50 ng, or about 100 ng, or about 200 ng, or about 300 ng,or about 400 ng, or about 500 ng, or about 600 ng, or about 700 ng, orabout 800 ng, or about 900 ng, or about 1000 ng, or about 1100 ng, orabout 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, orabout 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, orabout 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000 ng.

Some embodiments are directed to synthetic RNA molecules with lowtoxicity and high translation efficiency. Other embodiments are directedto a cell-culture medium for high-efficiency in vivo transfection,reprogramming, and gene editing of cells. Other embodiments pertain tomethods for producing synthetic RNA molecules encoding reprogrammingproteins. Still further embodiments pertain to methods for producingsynthetic RNA molecules encoding gene-editing proteins.

Some embodiments are directed to methods of gene-editing and/or genecorrection. Some embodiments encompass synthetic RNA-based gene-editingand/or gene correction, e.g. with RNA comprising non-canonicalnucleotides, e.g. RNA encoding one or more of a nuclease, atranscription activator-like effector nuclease (TALEN), a zinc-fingernuclease, a meganuclease, a nickase, a clustered regularly interspacedshort palindromic repeat (CRISPR)-associated protein a DNA-repairprotein, a DNA-modification protein, a base-modification protein, a DNAmethyltransferase, an protein that causes DNA demethylation, an enzymefor which DNA is a substrate or a natural or engineered variant,family-member, orthologue, fragment or fusion construct thereof. In someembodiments, the efficiency of the gene-editing and/or gene correctionis high, for example, higher than DNA-based gene editing and/or genecorrection. In some embodiments, the present methods of gene-editingand/or gene correction are efficient enough for in vivo application. Insome embodiments, the present methods of gene-editing and/or genecorrection are efficient enough to not require cellular selection (e.g.selection of cells that have been edited). In various embodiments, theefficiency of gene-editing of the present methods is about 1%, or about2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, orabout 8%, or about 9%, or about 10%, or about 20%, or about 30%, orabout 40%, or about 50%, or about 60%, or about 70%, or about 80%, orabout 90%, or about 100%. In various embodiments, the efficiency ofgene-correction of the present methods is about 1%, or about 2%, orabout 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%,or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, orabout 100%

Some embodiments are directed to high-efficiency gene-editing proteinscomprising engineered nuclease cleavage or DNA-modification domains.Other embodiments are directed to high-fidelity gene-editing proteinscomprising engineered nuclease cleavage or DNA-modification domains.Various embodiments are directed to high-efficiency gene-editingproteins comprising engineered DNA-binding domains. Other embodimentsare directed to high-fidelity gene-editing proteins comprisingengineered DNA-binding domains. Still other embodiments are directed togene-editing proteins comprising engineered repeat sequences. Someembodiments are directed to gene-editing proteins comprising one or moreCRISPR associated family members. Some embodiments are directed tomethods for altering the DNA sequence of a cell by transfecting the cellwith or inducing the cell to express a gene-editing protein. Otherembodiments are directed to methods for altering the DNA sequence of acell that is present in an in vitro culture. Still further embodimentsare directed to methods for altering the DNA sequence of a cell that ispresent in vivo.

Some embodiments are directed to methods of modulating the secretion orsubcellular localization of a polypeptide. In such embodiments, thepresent invention provides an RNA encoding a protein comprising a signalpeptide operably linked to a polypeptide having biologicalfunctionality. In an embodiment, the signal peptide modulates thesecretion of the polypeptide. In another embodiment, the signal peptidemodulates the subcellular localization of the polypeptide. For example,BMP7 comprising at least one FGF21 signal peptide may result inincreased secretion of BMP7. In another example, BMP7 comprising atleast one FGF21 signal peptide in place of the endogenous BMP7 signalpeptide may result in increased secretion of BMP7. In variousembodiments, any of the proteins listed in Table 2A or Table 2Bcomprising at least one signal peptide listed in the table below mayresult in increased secretion of the proteins.

SEQ ID Protein Signal Peptide NO Gaussia luciferase MGVKVLFALICIAVAEA596 Human BMP7 MHVRSLRAAAPHSFVALWAPLFLLRSALA 597Human chymotrypsinogen B MAFLWLLSCWALLGTTFG 598 Human chymotrypsinogen CMLGITVLAALLACASS 599 Human EPO MGVHECPAWLWLLLSLLSLPLGLPVLG 600Human FGF19 MRSGCVVVHVWILAGLWLAVAGRP 601 Human FGF21MDSDETGFEHSGLWVSVLAGLLLGACQA 602 Human FGF23 MLGARLRLWVCALCSVCSMSVLRA603 Human IL2 MYRMQLLSCIALSLALVTNS 604 Human IL22MAALQKSVSSFLMGTLATSCLLLLALLVQGGAA 605 Human IL6MNSFSTSAFGPVAFSLGLLLVLPAAFPAP 606 Human Interferon Alpha-2MALTFALLVALLVLSCKSSCSVG 607 Human Interferon Beta MTNKCLLQIALLLCFSTTALS608 Human Interferon Gamma MKYTSYILAFQLCIVLGSLGCYC 609 Human Trypsin-1MNPLLILTFVAAALA 610

Some embodiments are directed to methods of modulating the serumhalf-life, secretion, bioavailability, and/or activity of a proteincomprising administering a RNA encoding the protein and a RNA encoding areceptor for the protein. For example, IL15 may be administered with itsreceptor, e.g., IL15RA to enhance one or more of the serum half-life,secretion, bioavailability, and activity of IL15.

Many proteins and peptides, when translated from in vitro transcribedRNA, can exhibit reduced activity resulting from incomplete orinadequate post-translational processing. It has now been discoveredthat the amount of active protein, polypeptide or peptide producedfollowing RNA transfection can be increased by contacting cells withand/or inducing cells to express a second protein that is capable ofprocessing a polypeptide into the protein, peptide or polypeptide.Certain embodiments are therefore directed to a method for inducing acell to express an active protein, polypeptide or peptide comprisingcontacting the cell with a synthetic RNA molecule encoding a firstpolypeptide and contacting the cell with a second protein that iscapable of processing the first polypeptide into an active protein,polypeptide or peptide.

In certain embodiments, the second protein is administered to a patient.In one embodiment, the second protein is a recombinant protein. Inanother embodiment, the cells are contacted with a synthetic RNAmolecule encoding the second protein. In a further embodiment, the cellsare contacted with and/or induced to express an inhibitor of a moleculethat inhibits the second protein. In one embodiment, the inhibitor is ashort interfering RNA molecule.

In certain embodiments, the second protein is a member of the PCSKfamily. In other embodiments, the second protein is a proproteinconvertase. In still other embodiments, the second protein is aprohormone convertase. In still other embodiments, the second protein isa carboxypeptidase. In one embodiment, the second protein is PCSK3(Furin/PACE). In another embodiment, the second protein is primarilysecreted. In yet another embodiment, the second protein is primarilyintracellular. In a further embodiment, the first polypeptide, protein,polypeptide or peptide is selected from the group: a product ofproopiomelanocortin, renin, a product of enkephalin, a product ofprodynorphin, somatostatin, insulin, agouti-related peptide, glucagon,parathyroid hormone, a member of the transforming growth factor betasuperfamily, albumin, beta-secretase 1, nerve growth factor, caldesmon,the alpha-integrins, factor IX, α-melanocyte-stimulating hormone,adrenocorticotropic hormone, β-endorphin, and met-enkefalin.

Glycation and glycosylation are processes by which one or more sugarmolecules are bound to a protein. It has now been discovered thataltering the number or location of glycation and glycosylation sites canincrease or decrease the stability of a protein. Certain embodiments aretherefore directed to a protein with one or more glycation orglycosylation sites. In one embodiment, the protein is engineered tohave more glycation or glycosylation sites than a natural variant of theprotein. In another embodiment, the protein is engineered to have fewerglycation or glycosylation sites than a natural variant of the protein.In yet another embodiment, the protein has increased stability. In yetanother embodiment, the protein has decreased stability. In someembodiments, the protein is a circulating protein. In one embodiment,the protein is erythropoietin or a biologically active fragment,variant, analogue, or family-member thereof. In another embodiment, theprotein is darbepoetin alfa or a biologically active fragment, variant,analogue, or family-member thereof. In another embodiment, the proteinis NOVEPOETIN or a biologically active fragment, variant, analogue, orfamily-member thereof.

It has been further discovered that in certain situations, including oneor more steroids and/or one or more antioxidants in the transfectionmedium can increase in vivo transfection efficiency, in vivoreprogramming efficiency, and in vivo gene-editing efficiency. Certainembodiments are therefore directed to contacting a cell or patient witha glucocorticoid, such as hydrocortisone, prednisone, prednisolone,methylprednisolone, dexamethasone or betamethasone. Other embodimentsare directed to a method for inducing a cell to express a protein ofinterest by contacting a cell with a medium containing a steroid andcontacting the cell with one or more nucleic acid molecules. In oneembodiment, the nucleic acid molecule comprises synthetic RNA. Inanother embodiment, the steroid is hydrocortisone. In yet anotherembodiment, the hydrocortisone is present in the medium at aconcentration of between about 0.1 uM and about 10 uM, or about 1 uM.Other embodiments are directed to a method for inducing a cell in vivoto express a protein of interest by contacting the cell with a mediumcontaining an antioxidant and contacting the cell with one or morenucleic acid molecules. In one embodiment, the antioxidant is ascorbicacid or ascorbic-acid-2-phosphate. In another embodiment, the ascorbicacid or ascorbic-acid-2-phosphate is present in the medium at aconcentration of between about 0.5 mg/L and about 500 mg/L, includingabout 50 mg/L. Still other embodiments are directed to a method forreprogramming and/or gene-editing a cell in vivo by contacting the cellwith a medium containing a steroid and/or an antioxidant and contactingthe cell with one or more nucleic acid molecules, wherein the one ormore nucleic acid molecules encodes one or more reprogramming and/orgene-editing proteins. In certain embodiments, the cell is present in anorganism, and the steroid and/or antioxidant are delivered to theorganism.

Adding transferrin to the complexation medium has been reported toincrease the efficiency of plasmid transfection in certain situations.It has now been discovered that adding transferrin to the complexationmedium can also increase the efficiency of in vivo transfection withsynthetic RNA molecules. Certain embodiments are therefore directed to amethod for inducing a cell in vivo to express a protein of interest byadding one or more synthetic RNA molecules and a transfection reagent toa solution containing transferrin. In one embodiment, the transferrin ispresent in the solution at a concentration of between about 1 mg/L andabout 100 mg/L, such as about 5 mg/L. In another embodiment, thetransferrin is recombinant.

In certain situations, including pertaining to culturing, it may bedesirable to replace animal-derived components with non-animal-derivedand/or recombinant components, in part because non-animal-derived and/orrecombinant components can be produced with a higher degree ofconsistency than animal-derived components, and in part becausenon-animal-derived and/or recombinant components carry less risk ofcontamination with toxic and/or pathogenic substances than doanimal-derived components. Certain embodiments are therefore directed toa protein that is non-animal-derived and/or recombinant. Otherembodiments are directed to a medium, wherein some or all of thecomponents of the medium are non-animal-derived and/or recombinant.

Other embodiments are directed to a method for transfecting a cell invivo. In one embodiment, a cell in vivo is transfected with one or morenucleic acids, and the transfection is performed using a transfectionreagent, such as a lipid-based transfection reagent. In one embodiment,the one or more nucleic acids includes at least one RNA molecule. Inanother embodiment, the cell is transfected with one or more nucleicacids, and the one or more nucleic acids encodes at least one of: p53,TERT, an antibody, an extracellular matrix protein, a cytokine, asecreted protein, a membrane-bound protein, an enzyme, a gene-editingprotein, a chromatin-modifying protein, a DNA-binding protein, atranscription factor, a histone deacetylase, a pathogen-associatedmolecular pattern, and a tumor-associated antigen or a biologicallyactive fragment, analogue, variant or family-member thereof. In anotherembodiment, the cell is transfected repeatedly, such as at least about 2times during about 10 consecutive days, or at least about 3 times duringabout 7 consecutive days, or at least about 4 times during about 6consecutive days. Some embodiments are directed to a method forincreasing expression of telomerase in one of a fibroblast, ahematopoietic stem cell, a mesenchymal stem cells, a cardiac stem cell,a hair follicle stem cell, a neural stem cell, an intestinal stem cell,an endothelial stem cell, an olfactory stem cell, a neural crest stemcell, a testicular cell, and a keratinocyte. Some embodiments aredirected to a method for increasing the length of telomeres in one of afibroblast, a hematopoietic stem cell, a mesenchymal stem cells, acardiac stem cell, a hair follicle stem cell, a neural stem cell, anintestinal stem cell, an endothelial stem cell, an olfactory stem cell,a neural crest stem cell, a testicular cell, and a keratinocyte. Otherembodiments are directed to a method for isolating a cell from apatient, contacting the cell with a nucleic acid drug encoding acomponent of telomerase (e.g., TERT), and reintroducing the cell to thepatient. Various embodiments are directed to a method for increasing thereplicative potential of a cell.

Reprogramming can be performed by transfecting cells with one or morenucleic acids encoding one or more reprogramming factors. Examples ofreprogramming factors include, but are not limited to: Oct4 protein,Sox2 protein, Klf4 protein, c-Myc protein, I-Myc protein, TERT protein,Nanog protein, Lin28 protein, Utf1 protein, Aicda protein, miR200micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA andbiologically active fragments, analogues, variants and family-membersthereof. Certain embodiments are therefore directed to a method forreprogramming a cell in vivo. In one embodiment, the cell in vivo isreprogrammed by transfecting the cell with one or more nucleic acidsencoding one or more reprogramming factors. In one embodiment, the oneor more nucleic acids includes an RNA molecule that encodes Oct4protein. In another embodiment, the one or more nucleic acids alsoincludes one or more RNA molecules that encodes Sox2 protein, Klf4protein, and c-Myc protein. In yet another embodiment, the one or morenucleic acids also includes an RNA molecule that encodes Lin28 protein.In one embodiment, the cell is a human skin cell, and the human skincell is reprogrammed to a pluripotent stem cell. In another embodiment,the cell is a human skin cell, and the human skin cell is reprogrammedto a glucose-responsive insulin-producing cell. Examples of other cellsthat can be reprogrammed and other cells to which a cell can bereprogrammed include, but are not limited to: skin cells, pluripotentstem cells, mesenchymal stem cells, β-cells, retinal pigmentedepithelial cells, hematopoietic cells, cardiac cells, airway epithelialcells, neural stem cells, neurons, glial cells, bone cells, blood cells,and dental pulp stem cells. In one embodiment, the cell is contactedwith a medium that supports the reprogrammed cell. In one embodiment,the medium also supports the cell.

Importantly, infecting skin cells with viruses encoding Oct4, Sox2,Klf4, and c-Myc, combined with culturing the cells in a medium thatsupports the growth of cardiomyocytes, has been reported to causereprogramming of the skin cells to cardiomyocytes, without firstreprogramming the skin cells to pluripotent stem cells (See Efs et alNat Cell Biol. 2011; 13:215-22, the contents of which are herebyincorporated by reference). In certain situations, direct reprogramming(reprogramming one somatic cell to another somatic cell without firstreprogramming the somatic cell to a pluripotent stem cell, also known as“transdifferentiation”) may be desirable, in part because culturingpluripotent stem cells can be time-consuming and expensive, theadditional handling involved in establishing and characterizing a stablepluripotent stem cell line can carry an increased risk of contamination,and the additional time in culture associated with first producingpluripotent stem cells can carry an increased risk of genomicinstability and the acquisition of mutations, including point mutations,copy-number variations, and karyotypic abnormalities. Certainembodiments are therefore directed to a method for reprogramming asomatic cell in vivo, wherein the cell is reprogrammed to a somaticcell, and wherein a characterized pluripotent stem-cell line is notproduced.

It has been further discovered that, in certain situations, fewer totaltransfections may be required to reprogram a cell according to themethods of the present invention than according to other methods.Certain embodiments are therefore directed to a method for reprogramminga cell in vivo, wherein between about 1 and about 12 transfections areperformed during about 20 consecutive days, or between about 4 and about10 transfections are performed during about 15 consecutive days, orbetween about 4 and about 8 transfections are performed during about 10consecutive days. It is recognized that when a cell is contacted with amedium containing nucleic acid molecules, the cell may likely come intocontact with and/or internalize more than one nucleic acid moleculeeither simultaneously or at different times. A cell can therefore becontacted with a nucleic acid more than once, e.g. repeatedly, even whena cell is contacted only once with a medium containing nucleic acids.

Of note, nucleic acids can contain one or more non-canonical or“modified” residues as described herein. For instance, any of thenon-canonical nucleotides described herein can be used in the presentreprogramming methods. In one embodiment, pseudouridine-5′-triphosphatecan be substituted for uridine-5′-triphosphate in an invitro-transcription reaction to yield synthetic RNA, wherein up to 100%of the uridine residues of the synthetic RNA may be replaced withpseudouridine residues. In vitro-transcription can yield RNA withresidual immunogenicity, even when pseudouridine and 5-methylcytidineare completely substituted for uridine and cytidine, respectively (see,e.g., Angel. Reprogramming Human Somatic Cells to Pluripotency Using RNA[Doctoral Thesis]. Cambridge, Mass.: MIT; 2011, the contents of whichare hereby incorporated by reference). For this reason, it is common toadd an immunosuppressant to the transfection medium when transfectingcells with RNA. In certain situations, adding an immunosuppressant tothe transfection medium may not be desirable, in part because therecombinant immunosuppressant most commonly used for this purpose, B18R,can be expensive and difficult to manufacture. It has now beendiscovered that cells in vivo can be transfected and/or reprogrammedaccording to the methods of the present invention, without using B18R orany other immunosuppressant. It has been further discovered thatreprogramming cells in vivo according to the methods of the presentinvention without using immunosuppressants can be rapid, efficient, andreliable. Certain embodiments are therefore directed to a method fortransfecting a cell in vivo, wherein the transfection medium does notcontain an immunosuppressant. Other embodiments are directed to a methodfor reprogramming a cell in vivo, wherein the transfection medium doesnot contain an immunosuppressant. In certain situations, for examplewhen using a high cell density, it may be beneficial to add animmunosuppressant to the transfection medium. Certain embodiments aretherefore directed to a method for transfecting a cell in vivo, whereinthe transfection medium contains an immunosuppressant. Other embodimentsare directed to a method for reprogramming a cell in vivo, wherein thetransfection medium contains an immunosuppressant. In one embodiment,the immunosuppressant is B18R or a biologically active fragment,analogue, variant or family-member thereof or dexamethasone or aderivative thereof. In one embodiment, the transfection medium does notcontain an immunosuppressant, and the nucleic-acid dose is chosen toprevent excessive toxicity. In another embodiment, the nucleic-acid doseis less than about 1 mg/cm² of tissue or less than about 1 mg/100,000cells or less than about 10 mg/kg.

Reprogrammed cells produced according to certain embodiments of thepresent invention are suitable for therapeutic and/or cosmeticapplications as they do not contain undesirable exogenous DNA sequences,and they are not exposed to animal-derived or human-derived products,which may be undefined, and which may contain toxic and/or pathogeniccontaminants. Furthermore, the high speed, efficiency, and reliabilityof certain embodiments of the present invention may reduce the risk ofacquisition and accumulation of mutations and other chromosomalabnormalities. Certain embodiments of the present invention can thus beused to generate cells that have a safety profile adequate for use intherapeutic and/or cosmetic applications. For example, reprogrammingcells using RNA and the medium of the present invention, wherein themedium does not contain animal or human-derived components, can yieldcells that have not been exposed to allogeneic material. Certainembodiments are therefore directed to a reprogrammed cell that has adesirable safety profile. In one embodiment, the reprogrammed cell has anormal karyotype. In another embodiment, the reprogrammed cell has fewerthan about 5 copy-number variations (CNVs) relative to the patientgenome, such as fewer than about 3 copy-number variations relative tothe patient genome, or no copy-number variations relative to the patientgenome. In yet another embodiment, the reprogrammed cell has a normalkaryotype and fewer than about 100 single nucleotide variants in codingregions relative to the patient genome, or fewer than about 50 singlenucleotide variants in coding regions relative to the patient genome, orfewer than about 10 single nucleotide variants in coding regionsrelative to the patient genome.

Endotoxins and nucleases can co-purify and/or become associated withother proteins, such as serum albumin. Recombinant proteins, inparticular, can often have high levels of associated endotoxins andnucleases, due in part to the lysis of cells that can take place duringtheir production. Endotoxins and nucleases can be reduced, removed,replaced or otherwise inactivated by many of the methods of the presentinvention, including, for example, by acetylation, by addition of astabilizer such as sodium octanoate, followed by heat treatment, by theaddition of nuclease inhibitors to the albumin solution and/or medium,by crystallization, by contacting with one or more ion-exchange resins,by contacting with charcoal, by preparative electrophoresis or byaffinity chromatography. It has now been discovered that partially orcompletely reducing, removing, replacing or otherwise inactivatingendotoxins and/or nucleases from a medium and/or from one or morecomponents of a medium can increase the efficiency with which cells canbe transfected and reprogrammed. Certain embodiments are thereforedirected to a method for transfecting a cell in vivo with one or morenucleic acids, wherein the transfection medium is treated to partiallyor completely reduce, remove, replace or otherwise inactivate one ormore endotoxins and/or nucleases. Other embodiments are directed to amedium that causes minimal degradation of nucleic acids. In oneembodiment, the medium contains less than about 1 EU/mL, or less thanabout 0.1 EU/mL, or less than about 0.01 EU/mL.

In certain situations, protein-based lipid carriers such as serumalbumin can be replaced with non-protein-based lipid carriers such asmethyl-beta-cyclodextrin. The medium of the present invention can alsobe used without a lipid carrier, for example, when transfection isperformed using a method that may not require or may not benefit fromthe presence of a lipid carrier, for example, using one or morelipid-based transfection reagents, polymer-based transfection reagentsor peptide-based transfection reagents or using electroporation. Manyprotein-associated molecules, such as metals, can be highly toxic tocells in vivo. This toxicity can cause decreased viability, as well asthe acquisition of mutations. Certain embodiments thus have theadditional benefit of producing cells that are free from toxicmolecules.

The associated-molecule component of a protein can be measured bysuspending the protein in solution and measuring the conductivity of thesolution. Certain embodiments are therefore directed to a medium thatcontains a protein, wherein about a 10% solution of the protein in waterhas a conductivity of less than about 500 μmho/cm. In one embodiment,the solution has a conductivity of less than about 50 μmho/cm. Inanother embodiment, less than about 0.65% of the dry weight of theprotein comprises lipids and/or less than about 0.35% of the dry weightof the protein comprises free fatty acids.

The amount of nucleic acid delivered to cells in vivo can be increasedto increase the desired effect of the nucleic acid. However, increasingthe amount of nucleic acid delivered to cells in vivo beyond a certainpoint can cause a decrease in the viability of the cells, due in part totoxicity of the transfection reagent. It has now been discovered thatwhen a nucleic acid is delivered to a population of cells in vivo in afixed volume (for example, cells in a region of tissue), the amount ofnucleic acid delivered to each cell can depend on the total amount ofnucleic acid delivered to the population of cells and to the density ofthe cells, with a higher cell density resulting in less nucleic acidbeing delivered to each cell. In certain embodiments, a cell in vivo istransfected with one or more nucleic acids more than once. Under certainconditions, for example when the cells are proliferating, the celldensity may change from one transfection to the next. Certainembodiments are therefore directed to a method for transfecting a cellin vivo with a nucleic acid, wherein the cell is transfected more thanonce, and wherein the amount of nucleic acid delivered to the cell isdifferent for two of the transfections. In one embodiment, the cellproliferates between two of the transfections, and the amount of nucleicacid delivered to the cell is greater for the second of the twotransfections than for the first of the two transfections. In anotherembodiment, the cell is transfected more than twice, and the amount ofnucleic acid delivered to the cell is greater for the second of threetransfections than for the first of the same three transfections, andthe amount of nucleic acid delivered to the cells is greater for thethird of the same three transfections than for the second of the samethree transfections. In yet another embodiment, the cell is transfectedmore than once, and the maximum amount of nucleic acid delivered to thecell during each transfection is sufficiently low to yield at leastabout 80% viability for at least two consecutive transfections.

It has now been discovered that modulating the amount of nucleic aciddelivered to a population of proliferating cells in vivo in a series oftransfections can result in both an increased effect of the nucleic acidand increased viability of the cells. It has been further discoveredthat, in certain situations, when cells in vivo are contacted with oneor more nucleic acids encoding one or more reprogramming factors in aseries of transfections, the efficiency of reprogramming can beincreased when the amount of nucleic acid delivered in latertransfections is greater than the amount of nucleic acid delivered inearlier transfections, for at least part of the series of transfections.Certain embodiments are therefore directed to a method for reprogramminga cell in vivo, wherein one or more nucleic acids is repeatedlydelivered to the cell in a series of transfections, and the amount ofthe nucleic acid delivered to the cell is greater for at least one latertransfection than for at least one earlier transfection. In oneembodiment, the cell is transfected between about 2 and about 10 times,or between about 3 and about 8 times, or between about 4 and about 6times. In another embodiment, the one or more nucleic acids includes atleast one RNA molecule, the cell is transfected between about 2 andabout 10 times, and the amount of nucleic acid delivered to the cell ineach transfection is the same as or greater than the amount of nucleicacid delivered to the cell in the most recent previous transfection. Inyet another embodiment, the amount of nucleic acid delivered to the cellin the first transfection is between about 20 ng/cm² and about 250ng/cm², or between 100 ng/cm² and 600 ng/cm². In yet another embodiment,the cell is transfected about 5 times at intervals of between about 12and about 48 hours, and the amount of nucleic acid delivered to the cellis about 25 ng/cm² for the first transfection, about 50 ng/cm² for thesecond transfection, about 100 ng/cm² for the third transfection, about200 ng/cm² for the fourth transfection, and about 400 ng/cm² for thefifth transfection. In yet another embodiment, the cell is furthertransfected at least once after the fifth transfection, and the amountof nucleic acid delivered to the cell is about 400 ng/cm².

Certain embodiments are directed to a method for transfecting a cell invivo with a nucleic acid, wherein the amount of nucleic acid isdetermined by measuring the cell density, and choosing the amount ofnucleic acid to transfect based on the measurement of cell density. Inone embodiment, the cell density is measured by optical means. Inanother embodiment, the cell is transfected repeatedly, the cell densityincreases between two transfections, and the amount of nucleic acidtransfected is greater for the second of the two transfections than forthe first of the two transfections.

It has now been discovered that, in certain situations, the amount of acirculating protein that is produced in a patient can be increased byadministering to a patient a nucleic acid at a plurality ofadministration sites. In certain embodiments, the amount of acirculating protein is increased relative to the amount of thecirculating protein that is produced in a patient by administering tothe patient the nucleic acid at a single injection site. In oneembodiment, the administering is by injection. In another embodiment,the injection is intradermal injection. In still another embodiment, theinjection is subcutaneous or intramuscular injection. In someembodiments, the plurality of administration sites compriseadministration sites in the skin. In other embodiments, the plurality ofadministration sites are at least about 1 or at least about 2 or atleast about 5 or at least about 10 or at least about 20 or at leastabout 50 or at least about 100 administration sites. In one embodiment,the administering is performed within at least about 5 minutes or atleast about 10 minutes or at least about 30 minutes or at least about 1hour or at least about 2 hours or at least about 5 hours or at leastabout 12 hours or at least about 1 day. In certain embodiments, theamount of a circulating protein is increased by at least about 10percent or at least about 20 percent or at least about 50 percent or atleast about 100 percent or at least about 3-fold or at least about5-fold or at least about 10-fold or at least about 20-fold or at leastabout 50-fold or at least about 100-fold or at least about 500-fold orat least about 1000-fold or greater than 1000-fold.

It has now been discovered that, in certain situations, the in vivotransfection efficiency and viability of cells contacted with the mediumof the present invention can be improved by conditioning the medium.Certain embodiments are therefore directed to a method for conditioninga medium. Other embodiments are directed to a medium that isconditioned. In one embodiment, the feeders are fibroblasts, and themedium is conditioned for approximately 24 hours. Other embodiments aredirected to a method for transfecting a cell in vivo, wherein thetransfection medium is conditioned. Other embodiments are directed to amethod for reprogramming and/or gene-editing a cell in vivo, wherein themedium is conditioned. In one embodiment, the feeders are mitoticallyinactivated, for example, by exposure to a chemical such as mitomycin-Cor by exposure to gamma radiation. In certain embodiments, it may bebeneficial to use only autologous materials, in part to, for example andnot wishing to be bound by theory, avoid the risk of diseasetransmission from the feeders to the cell or the patient. Certainembodiments are therefore directed to a method for transfecting a cellin vivo, wherein the transfection medium is conditioned, and wherein thefeeders are derived from the same individual as the cell beingtransfected. Other embodiments are directed to a method forreprogramming and/or gene-editing a cell in vivo, wherein the medium isconditioned, and wherein the feeders are derived from the sameindividual as the cell being reprogrammed and/or gene-edited.

Several molecules can be added to media by conditioning. Certainembodiments are therefore directed to a medium that is supplemented withone or more molecules that are present in a conditioned medium. In oneembodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or abiologically active fragment, analogue, variant, agonist, orfamily-member thereof. In another embodiment, the medium is supplementedwith TGF-β or a biologically active fragment, analogue, variant,agonist, or family-member thereof. In yet another embodiment, a cell invivo is reprogrammed according to the method of the present invention,wherein the medium is not supplemented with TGF-β for between about 1and about 5 days, and is then supplemented with TGF-β for at least about2 days. In yet another embodiment, the medium is supplemented with IL-6,IL-6R or a biologically active fragment, analogue, variant, agonist, orfamily-member thereof. In yet another embodiment, the medium issupplemented with a sphingolipid or a fatty acid. In still anotherembodiment, the sphingolipid is lysophosphatidic acid,lysosphingomyelin, sphingosine-1-phosphate or a biologically activeanalogue, variant or derivative thereof.

In addition to mitotically inactivating cells, under certain conditions,irradiation can change the gene expression of cells, causing cells toproduce less of certain proteins and more of certain other proteins thatnon-irradiated cells, for example, members of the Wnt family ofproteins. In addition, certain members of the Wnt family of proteins canpromote the growth and transformation of cells. It has now beendiscovered that, in certain situations, the efficiency of reprogrammingcan be greatly increased by contacting a cell in vivo with a medium thatis conditioned using irradiated feeders instead of mitomycin-c-treatedfeeders. It has been further discovered that the increase inreprogramming efficiency observed when using irradiated feeders iscaused in part by Wnt proteins that are secreted by the feeders. Certainembodiments are therefore directed to a method for reprogramming a cellin vivo, wherein the cell is contacted with Wnt1, Wnt2, Wnt3, Wnt3a or abiologically active fragment, analogue, variant, family-member oragonist thereof, including agonists of downstream targets of Wntproteins, and/or agents that mimic one or more of the biological effectsof Wnt proteins, for example,2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

Because of the low efficiency of many DNA-based reprogramming methods,these methods may be difficult or impossible to use with cells derivedfrom patient samples, which may contain only a small number of cells. Incontrast, the high efficiency of certain embodiments of the presentinvention can allow reliable reprogramming of a small number of cells,including single cells. Certain embodiments are directed to a method forreprogramming a small number of cells. Other embodiments are directed toa method for reprogramming a single cell. In one embodiment, the cell iscontacted with one or more enzymes. In another embodiment, the enzyme iscollagenase. In yet another embodiment, the collagenase isanimal-component free. In one embodiment, the collagenase is present ata concentration of between about 0.1 mg/mL and about 10 mg/mL, orbetween about 0.5 mg/mL and about 5 mg/mL. In another embodiment, thecell is a blood cell. In yet another embodiment, the cell is contactedwith a medium containing one or more proteins that is derived from thepatient's blood. In still another embodiment, the cell is contacted witha medium comprising: DMEM/F12+2 mM L-alanyl-L-glutamine+between about 5%and about 25% patient-derived serum, or between about 10% and about 20%patient-derived serum, or about 20% patient-derived serum.

It has now been discovered that, in certain situations, transfectingcells in vivo with a mixture of RNA encoding Oct4, Sox2, Klf4, and c-Mycusing the medium of the present invention can cause the rate ofproliferation of the cells to increase. When the amount of RNA deliveredto the cells is too low to ensure that all of the cells are transfected,only a fraction of the cells may show an increased proliferation rate.In certain situations, such as when generating a personalizedtherapeutic, increasing the proliferation rate of cells may bedesirable, in part because doing so can reduce the time necessary togenerate the therapeutic, and therefore can reduce the cost of thetherapeutic. Certain embodiments are therefore directed to a method fortransfecting a cell in vivo with a mixture of RNA encoding Oct4, Sox2,Klf4, and c-Myc. In one embodiment, the cell exhibits an increasedproliferation rate. In another embodiment, the cell is reprogrammed.

Many diseases are associated with one or more mutations. Mutations canbe corrected by contacting a cell with a nucleic acid that encodes aprotein that, either alone or in combination with other molecules,corrects the mutation (an example of gene-editing). Examples of suchproteins include: a nuclease, a transcription activator-like effectornuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, aclustered regularly interspaced short palindromic repeat(CRISPR)-associated protein a DNA-repair protein, a DNA-modificationprotein, a base-modification protein, a DNA methyltransferase, anprotein that causes DNA demethylation, an enzyme for which DNA is asubstrate or a natural or engineered variant, family-member, orthologue,fragment or fusion construct thereof. Certain embodiments are thereforedirected to a method for transfecting a cell in vivo with a nucleicacid, wherein the nucleic acid encodes a protein that, either alone orin combination with other molecules, creates a single-strand ordouble-strand break in a DNA molecule. In a one embodiment, the proteinis a zinc finger nuclease or a TALEN. In another embodiment, the nucleicacid is an RNA molecule. In yet another embodiment, the single-strand ordouble-strand break is within about 5,000,000 bases of the transcriptionstart site of a gene selected from the group: SERPINA1, CCR5, CXCR4,GAD1, GAD2, CFTR, HBA1, HBA2, HBB, HBD, FANCA, XPA, XPB, XPC, ERCC2,POLH, HTT, DMD, SOD1, APOE, PRNP, BRCA1, and BRCA2 or an analogue,variant or family-member thereof. In one embodiment, the presentinvention relates to gene-editing of the MYC protein (e.g. correctingone or more mutations that may be linked to cancer), optionally with aTALEN. In yet another embodiment, the cell is transfected with a nucleicacid that acts as a repair template by either causing the insertion of aDNA sequence in the region of the single-strand or double-strand breakor by causing the DNA sequence in the region of the single-strand ordouble-strand break to otherwise change. In yet another embodiment, thegene-editing protein contains a DNA modification domain. In yet anotherembodiment, the gene-editing protein corrects a mutation withoutcreating a single-strand break. In yet another embodiment, thegene-editing protein corrects a mutation without creating adouble-strand break. In yet another embodiment, the gene-editing proteincorrects a mutation by causing the replacement of one base with anotherbase. In one embodiment, adenine is replaced by cytosine. In anotherembodiment, adenine is replaced by guanine. In yet another embodiment,adenine is replaced by thymine. In yet another embodiment, cytosine isreplaced by adenine. In yet another embodiment, cytosine is replaced byguanine. In yet another embodiment, cytosine is replaced by thymine. Inyet another embodiment, guanine is replaced by adenine. In yet anotherembodiment, guanine is replaced by cytosine. In yet another embodiment,guanine is replaced by thymine. In yet another embodiment, thymine isreplaced by adenine. In yet another embodiment, thymine is replaced bycytosine. In yet another embodiment, thymine is replaced by guanine. Inone embodiment, the replacement of one base with another base is aone-step process. In another embodiment, the replacement of one basewith another base is a multi-step process. In some embodiments, one baseis replaced by more than one base, for example, by two bases. In otherembodiments, more than one base is replaced by one base. In still otherembodiments, more than one base is replaced by more than one base. Insome embodiments, the gene-editing protein contains a deaminase domain.In one embodiment the deaminase domain comprises a cytidine deaminasedomain. In another embodiment, the deaminase domain comprises anadenosine deaminase domain. In yet another embodiment, the deaminasedomain comprises a guanosine deaminase domain. In yet anotherembodiment, the gene-editing protein comprises a sequence that is atleast about 50% or at least about 60% or at least about 70% or at leastabout 80% or at least about 90% or at least about 95% or at least about99% homologous to one or more of: SEQ ID NOs: 587, 588, 589, 590, 591,592, and 593. In yet another embodiment, the gene-editing proteincomprises a linker. In one embodiment, the linker is a flexible linker.In another embodiment, the linker positions the deaminase domain inproximity to a target base. In yet another embodiment, the gene-editingprotein deaminates the target base. In yet another embodiment, thegene-editing protein comprises a glycosylase-inhibitor domain. In yetanother embodiment, the gene-editing protein comprisesglycosylase-inhibitor activity. In one embodiment, the glycosylaseinhibitor is a uracil glycosylase inhibitor. In another embodiment, theglycosylase inhibitor is a N-methylpurine DNA glycosylase inhibitor. Inyet another embodiment, the cell is reprogrammed, and subsequently, thecell is gene-edited. In yet another embodiment, the cell is gene-edited,and subsequently, the cell is reprogrammed. In yet another embodiment,the gene-editing and reprogramming are performed within about 7 days ofeach other. In yet another embodiment, the gene-editing andreprogramming occur simultaneously or on the same day. In yet anotherembodiment, the cell is a skin cell, the skin cell is gene-edited todisrupt the CCR5 gene, the skin cell is reprogrammed to a hematopoieticstem cell, thus producing a therapeutic for HIV/AIDS, and thetherapeutic is used to treat a patient with HIV/AIDS. In yet anotherembodiment, the skin cell is derived from the same patient whom thetherapeutic is used to treat.

Certain embodiments are directed to methods and compositions for thetreatment of rare diseases. In some embodiments, the rare disease is oneor more of: a rare metabolic disease, a rare cardiovascular disease, arare dermatologic disease, a rare neurologic disease, a raredevelopmental disease, a rare genetic disease, a rare pulmonary disease,a rare liver disease, a rare kidney disease, a rare psychiatric disease,a rare reproductive disease, a rare musculoskeletal disease, a rareorthopedic disease, an inborn error of metabolism, a lysosomal storagedisease, and a rare ophthalmologic disease. In one embodiment, thedisease is alpha-1-antitrypsin deficiency. Some embodiments are directedto a treatment comprising a nucleic acid encoding a gene-editing proteinthat is capable of causing a deletion in a gene that is associated withone or more of: a gain-of-function mutation, a loss-of-functionmutation, a recessive mutation, a dominant mutation or a dominantnegative mutation. Other embodiments are directed to a treatmentcomprising a nucleic acid encoding a gene-editing protein that iscapable of correcting one or more of: a gain-of-function mutation, aloss-of-function mutation, a recessive mutation, a dominant mutation ora dominant negative mutation. In one embodiment, the treatmentameliorates one or more of the symptoms in a subject. In anotherembodiment, the subject is a human subject. In yet another embodiment,the subject is a veterinary subject. Some embodiments are directed to atreatment for alpha-1-antitrypsin deficiency comprising administering toa subject a nucleic acid comprising a gene-editing protein that iscapable of causing a deletion in or near the SERPINA1 gene. Otherembodiments are directed to a treatment for alpha-1-antitrypsindeficiency comprising administering to a subject a nucleic acid encodinga gene-editing protein that is capable of correcting a mutation in ornear the SERPINA1 gene. In one embodiment the mutation is the Zmutation. In one embodiment, the deletion or correction reduces theaccumulation of polymerized alpha-1-antitrypsin protein in the subject'scells and/or increases the secretion of alpha-1-antitrypsin from thesubject's cells. In another embodiment, the treated cells regenerate adiseased organ. In yet another embodiment, the diseased organ is theliver. In yet another embodiment, the diseased organ is the lung. In yetanother embodiment, the treatment delays or eliminates the subject'sneed for a liver and/or lung transplant. Other embodiments are directedto a treatment for epidermolysis bullosa. In one embodiment theepidermolysis bullosa is dystrophic epidermolysis bullosa. In anotherembodiment, the epidermolysis bullosa is epidermolysis bullosa simplex.In yet another embodiment, the dystrophic epidermolysis bullosa isrecessive dystrophic epidermolysis bullosa. In some embodiments, thetreatment comprises administering to a subject a nucleic acid encoding agene-editing protein that is capable of correcting a mutation in or nearthe COL7A1 gene. In one embodiment, the correction increases the amountof functional collagen VII produced by the subject's cells. In anotherembodiment, the treatment reduces the size, severity, and/or frequencyof recurrence of skin lesions and/or blisters. Still other embodimentsare directed to a treatment for primary hyperoxaluria. In one embodimentthe primary hyperoxaluria is type I primary hyperoxaluria. In anotherembodiment, the treatment comprises administering to a subject a nucleicacid encoding a gene-editing protein that is capable of correcting amutation in or near the AGXT gene. In yet another embodiment, thetreatment delays or eliminates the subject's need for a kidney and/orliver transplant.

Genes that can be edited according to the methods of the presentinvention to produce therapeutics of the present invention include genesthat can be edited to restore normal function, as well as genes that canbe edited to reduce or eliminate function. Such genes include, but arenot limited to alpha-1-antitrypsin (SERPINA1), mutations in which cancause alpha-1-antitrypsin deficiency, beta globin (HBB), mutations inwhich can cause sickle cell disease (SCD) and β-thalassemia, breastcancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2),mutations in which can increase susceptibility to breast cancer, C-Cchemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4(CXCR4), mutations in which can confer resistance to HIV infection,cystic fibrosis transmembrane conductance regulator (CFTR), mutations inwhich can cause cystic fibrosis, dystrophin (DMD), mutations in whichcan cause muscular dystrophy, including Duchenne muscular dystrophy andBecker's muscular dystrophy, glutamate decarboxylase 1 and glutamatedecarboxylase 2 (GAD1, GAD2), mutations in which can prevent autoimmunedestruction of n-cells, hemoglobin alpha 1, hemoglobin alpha 2, andhemoglobin delta (HBA1, HBA2, and HBD), mutations in which can causethalassemia, desmoplakin, keratin 5, keratin 14, plectin, integrinalpha-6, integrin beta-4, laminin subunit alpha-3, laminin subunitbeta-3, laminin subunit gamma-2, collagen type VII alpha 1, collagentype XVII alpha 1, and matrix metalloproteinase-1 (DSP, KRT5, KRT14,PLEC1, ITGA6, ITGB4, LAMA3, LAMB3, LAMC2, COL7A1, COL17A1, and MMP1),mutations in which can cause epidermolysis bullosa, Huntington (HTT),mutations in which can cause Huntington's disease, superoxide dismutase1 (SOD1), mutations in which can cause amyotrophic lateral sclerosis(ALS), XPA, XPB, XPC, XPD (ERCC6) and polymerase (DNA directed), eta(POLH), mutations in which can cause xeroderma pigmentosum, leucine-richrepeat kinase 2 (LRRK2), mutations in which can cause Parkinson'sdisease, and Fanconi anemia, complementation groups A, B, C, D1, D2, E,F, G, I, J, L, M, N, P (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE,FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP), and RAD51homolog C (S. cerevisiae) (RAD51C), mutations in which can cause Fanconianemia.

In a specific embodiment, the present invention relates to method ofmodulating Growth-Factor-Differentiation-Factor-15 (GDF15). In aspecific embodiment, there is provided a method of treatment in which anRNA as described herein is administered to modulate GDF15 levels. Insome embodiments, the RNA encodes GDF15. In a specific embodiment, thegene that is edited is Growth-Factor-Differentiation-Factor-15 (GDF15).In various embodiments, the present invention relates to targetingand/or expressing GDF15 to treat or prevent a disease or disorder suchas a metabolic disease or disorder (e.g., diabetes (type I and II),insulin resistance, obesity, dyslipidemia, hypercholesterolemia,hyperglycemia, hyperinsulinemia, hypertension, hepatosteaotosis such asnon-alcoholic steatohepatitis (NASH) and non-alcoholic fatty acid liverdisease (NAFLD), cancer, a disease or disorder associated with impairedlipid metabolism, a disease or disorder associated with impaired renalfunction (e.g., chronic kidney diseases, nephropathy such as diabeticnephropathy, kidney failure), a disease or disorder associated withimpaired hepatic function, a disease or disorder associated withimpaired lung function, a vascular or cardiovascular disease or disorder(e.g., coronary artery disease, cardiomyopathy, hypertension, atrialfibrillation, preeclampsia, peripheral artery disease, atherosclerosis,heart failure, acute myocardial infarction, acute coronary syndrome,muscle wasting, hypertensive ventricular hypertrophy, hypertensivecardiomyopathy, ischemic heart disease, myocardial infarction, abdominalaortic aneurysm, a blood clot, deep vein thrombosis, venous stasisdisease, phlebitis, varicose veins etc.), muscle wasting, inflammation,and a respiratory disease. In a specific embodiment, targeting of GDF15improves lipid metabolism by mobilizing lipid deposits from the liverand/or other tissues into circulation for use in metabolism.

In certain disorders, the body may increase GDF15 levels in an attemptto counteract one or more negative aspects of the disease state. Certainembodiments are therefore directed to increasing GDF15 levels. In oneembodiment, the serum level of GDF15 is increased. In anotherembodiment, the level of GDF15 in a tissue and/or organ is increased. Inyet another embodiment, ALT levels are reduced. In yet anotherembodiment, AST levels are reduced. In still another embodiment, glucoselevels are reduced. In still another embodiment, serum cholesterollevels are increased. In still another embodiment, serum triglyceridelevels are increased. In yet another embodiment, food intake is reduced.In yet another embodiment, bodyweight is reduced. In a furtherembodiment, treatment improves adherence to a diet and/or exerciseregimen.

In a specific embodiment, expressing GDF15 improves lipid metabolism bymobilizing lipid deposits from the liver and/or other tissues intocirculation for use in metabolism. In a specific embodiment, expressingand/or targeting of GDF15 reduces liver inflammation. In a specificembodiment, expressing and/or targeting of GDF15 treats or prevents oneor more of fatty liver, NAFLD, NASH, inflammation, hepatitis, fibrosis,cirrhosis, and hepatocellular carcinoma. In a specific embodiment,expressing and/or targeting of GDF15 treats or prevents one or more ofchronic kidney disease, acute kidney injury, nephropathy, diabeticnephropathy, kidney failure, and kidney fibrosis. In a specificembodiment, expressing and/or targeting GDF15 in combination withexpressing and/or targeting another member of the TGF-β superfamilyimproves lipid metabolism by mobilizing lipid deposits from the liverand/or other tissues into circulation for use in metabolism, reducesliver inflammation, and/or treats or prevents one or more of fattyliver, NAFLD, NASH, inflammation, hepatitis, fibrosis, cirrhosis,hepatocellular carcinoma, chronic kidney disease, acute kidney injury,nephropathy, diabetic nephropathy, kidney failure, and kidney fibrosis.Certain embodiments are directed to a therapeutic comprising a nucleicacid. In one embodiment, the nucleic acid encodes one or moregene-editing proteins. Other embodiments are directed to a therapeuticcomprising one or more cells that are transfected, reprogrammed, and/orgene-edited in vivo according to the methods of the present invention.In one embodiment, a cell is transfected, reprogrammed, and/orgene-edited, and the transfected, reprogrammed, and/or gene-edited cellis introduced into a patient. In another embodiment, the cell isharvested from the same patient into whom the transfected, reprogrammedand/or gene-edited cell is introduced. Examples of diseases that can betreated with therapeutics of the present invention include, but are notlimited to Alzheimer's disease, spinal cord injury, amyotrophic lateralsclerosis, cystic fibrosis, heart disease, including ischemic anddilated cardiomyopathy, macular degeneration, Parkinson's disease,Huntington's disease, diabetes, sickle-cell anemia, thalassemia, Fanconianemia, xeroderma pigmentosum, muscular dystrophy, severe combinedimmunodeficiency, hereditary sensory neuropathy, cancer, and HIV/AIDS.In certain embodiments, the therapeutic comprises a cosmetic. In oneembodiment, a cell is harvested from a patient, the cell is reprogrammedand expanded to a large number of adipose cells to produce a cosmetic,and the cosmetic is introduced into the patient. In still anotherembodiment, the cosmetic is used for tissue reconstruction.

While detailed examples are provided herein for the production ofspecific types of cells and for the production of therapeuticscomprising specific types of cells, it is recognized that the methods ofthe present invention can be used to produce many other types of cells,and to produce therapeutics comprising one or more of many other typesof cells, for example, by reprogramming a cell according to the methodsof the present invention, and culturing the cell under conditions thatmimic one or more aspects of development by providing conditions thatresemble the conditions present in the cellular microenvironment duringdevelopment.

Certain embodiments are directed to a library of cells with a variety ofhuman leukocyte antigen (HLA) types (“HLA-matched libraries”). AnHLA-matched library may be beneficial in part because it can provide forthe rapid production and/or distribution of therapeutics without thepatient having to wait for a therapeutic to be produced from thepatient's cells. Such a library may be particularly beneficial for theproduction of cosmetics and for the treatment of heart disease anddiseases of the blood and/or immune system for which patients maybenefit from the immediate availability of a therapeutic or cosmetic.

The DNA sequence of a cell can be altered by contacting the cell with agene-editing protein or by inducing the cell to express a gene-editingprotein. However, previously disclosed gene-editing proteins suffer fromlow binding efficiency and excessive off-target activity, which canintroduce undesired mutations in the DNA of the cell, severely limitingtheir use in vivo, for example in therapeutic and cosmetic applications,in which the introduction of undesired mutations in a patient's cellscould lead to the development of cancer. It has now been discovered thatgene-editing proteins that comprise the StsI endonuclease cleavagedomain (SEQ ID NO: 1) can exhibit substantially lower off-targetactivity in vivo than previously disclosed gene-editing proteins, whilemaintaining a high level of on-target activity in vivo. Other novelengineered proteins have also been discovered that can exhibit highon-target activity in vivo, low off-target activity in vivo, small size,solubility, and other desirable characteristics when they are used asthe nuclease domain of a gene-editing protein: StsI-HA (SEQ ID NO: 2),StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO:5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7). StsI-HA,StsI-HA2 (high activity), StsI-UHA, and StsI-UHA2 (ultra-high activity)can exhibit higher on-target activity in vivo than both wild-type StsIand wild-type FokI, due in part to specific amino-acid substitutionswithin the N-terminal region at the 34 and 61 positions, while StsI-HF(high fidelity) and StsI-UHF (ultra-high fidelity) can exhibit loweroff-target activity in vivo than both wild-type StsI and wild-type FokI,due in part to specific amino-acid substitutions within the C-terminalregion at the 141 and 152 positions.

Certain embodiments are therefore directed to a protein. In someembodiments, the protein is present in vivo. In other embodiments, theprotein comprises a nuclease domain. In one embodiment, the nucleasedomain comprises one or more of: the cleavage domain of FokIendonuclease (SEQ ID NO: 53), the cleavage domain of StsI endonuclease(SEQ ID NO: 1), StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3),StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO:6), and StsI-UHF (SEQ ID NO: 7) or a biologically active fragment orvariant thereof.

It has also been discovered that engineered gene-editing proteins thatcomprise DNA-binding domains comprising certain novel repeat sequencescan exhibit lower off-target activity in vivo than previously disclosedgene-editing proteins, while maintaining a high level of on-targetactivity in vivo. Certain of these engineered gene-editing proteins canprovide several advantages over previously disclosed gene-editingproteins, including, for example, increased flexibility of the linkerregion connecting repeat sequences, which can result in increasedbinding efficiency. Certain embodiments are therefore directed to aprotein comprising a plurality of repeat sequences. In one embodiment,at least one of the repeat sequences contains the amino-acid sequence:GabG, where “a” and “b” each represent any amino acid. In oneembodiment, the protein is a gene-editing protein. In anotherembodiment, one or more of the repeat sequences are present in aDNA-binding domain. In a further embodiment, “a” and “b” are eachindependently selected from the group: H and G. In a still furtherembodiment, “a” and “b” are H and G, respectively. In one embodiment,the amino-acid sequence is present within about 5 amino acids of theC-terminus of the repeat sequence. In another embodiment, the amino-acidsequence is present at the C-terminus of the repeat sequence. In someembodiments, one or more G in the amino-acid sequence GabG is replacedwith one or more amino acids other than G, for example A, H or GG. Inone embodiment, the repeat sequence has a length of between about 32 andabout 40 amino acids or between about 33 and about 39 amino acids orbetween about 34 and 38 amino acids or between about 35 and about 37amino acids or about 36 amino acids or greater than about 32 amino acidsor greater than about 33 amino acids or greater than about 34 aminoacids or greater than about 35 amino acids. Other embodiments aredirected to a protein comprising one or more transcriptionactivator-like effector domains. In one embodiment, at least one of thetranscription activator-like effector domains comprises a repeatsequence. Other embodiments are directed to a protein comprising aplurality of repeat sequences generated by inserting one or more aminoacids between at least two of the repeat sequences of a transcriptionactivator-like effector domain. In one embodiment, one or more aminoacids is inserted about 1 or about 2 or about 3 or about 4 or about 5amino acids from the C-terminus of at least one repeat sequence. Stillother embodiments are directed to a protein comprising a plurality ofrepeat sequences, wherein about every other repeat sequence has adifferent length than the repeat sequence immediately preceding orfollowing the repeat sequence. In one embodiment, every other repeatsequence is about 36 amino acids long. In another embodiment, everyother repeat sequence is 36 amino acids long. Still other embodimentsare directed to a protein comprising a plurality of repeat sequences,wherein the plurality of repeat sequences comprises at least two repeatsequences that are each at least 36 amino acids long, and wherein atleast two of the repeat sequences that are at least 36 amino acids longare separated by at least one repeat sequence that is less than 36 aminoacids long. Some embodiments are directed to a protein that comprisesone or more sequences selected from, for example, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, andSEQ ID NO: 60.

Other embodiments are directed to a protein that comprises a DNA-bindingdomain. In some embodiments, the DNA-binding domain comprises aplurality of repeat sequences. In one embodiment, the plurality ofrepeat sequences enables high-specificity recognition of a binding sitein a target DNA molecule. In another embodiment, at least two of therepeat sequences have at least about 50%, or about 60%, or about 70%, orabout 80%, or about 90%, or about 95%, or about 98%, or about 99%homology to each other. In a further embodiment, at least one of therepeat sequences comprises one or more regions capable of binding to abinding site in a target DNA molecule. In a still further embodiment,the binding site comprises a defined sequence of between about 1 toabout 5 bases in length. In one embodiment, the DNA-binding domaincomprises a zinc finger. In another embodiment, the DNA-binding domaincomprises a transcription activator-like effector (TALE). In a furtherembodiment, the plurality of repeat sequences includes at least onerepeat sequence having at least about 50% or about 60% or about 70% orabout 80% or about 90% or about 95% or about 98%, or about 99% homologyto a TALE. In a still further embodiment, the gene-editing proteincomprises a clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein. In one embodiment, the gene-editing proteincomprises a nuclear-localization sequence. In another embodiment, thenuclear-localization sequence comprises the amino-acid sequence: PKKKRKV(SEQ ID NO: 471). In one embodiment, the gene-editing protein comprisesa mitochondrial-localization sequence. In another embodiment, themitochondrial-localization sequence comprises the amino-acid sequence:LGRVIPRKIASRASLM (SEQ ID NO: 472). In one embodiment, the gene-editingprotein comprises a linker. In another embodiment, the linker connects aDNA-binding domain to a nuclease domain. In a further embodiment, thelinker is between about 1 and about 10 amino acids long. In someembodiments, the linker is about 1, about 2, or about 3, or about 4, orabout 5, or about 6, or about 7, or about 8, or about 9, or about 10amino acids long. In one embodiment, the gene-editing protein is capableof generating a nick or a double-strand break in a target DNA molecule.

Certain embodiments are directed to a method for modifying the genome ofa cell in vivo, the method comprising introducing into a cell in vivo anucleic acid molecule encoding a non-naturally occurring fusion proteincomprising an artificial transcription activator-like (TAL) effectorrepeat domain comprising one or more repeat units 36 amino acids inlength and an endonuclease domain, wherein the repeat domain isengineered for recognition of a predetermined nucleotide sequence, andwherein the fusion protein recognizes the predetermined nucleotidesequence. In one embodiment, the cell is a eukaryotic cell. In anotherembodiment, the cell is an animal cell. In a further embodiment, thecell is a mammalian cell. In a still further embodiment, the cell is ahuman cell. In one embodiment, the cell is a plant cell. In anotherembodiment, the cell is a prokaryotic cell. In some embodiments, thefusion protein introduces an endonucleolytic cleavage in a nucleic acidof the cell, whereby the genome of the cell is modified.

Certain embodiments are directed to a composition for altering the DNAsequence of a cell in vivo comprising a nucleic acid, wherein thenucleic acid encodes a gene-editing protein. Other embodiments aredirected to a composition for altering the DNA sequence of a cell invivo comprising a nucleic-acid mixture, wherein the nucleic-acid mixturecomprises: a first nucleic acid that encodes a first gene-editingprotein, and a second nucleic acid that encodes a second gene-editingprotein. In one embodiment, the binding site of the first gene-editingprotein and the binding site of the second gene-editing protein arepresent in the same target DNA molecule. In another embodiment, thebinding site of the first gene-editing protein and the binding site ofthe second gene-editing protein are separated by less than about 50bases, or less than about 40 bases, or less than about 30 bases or lessthan about 20 bases, or less than about 10 bases, or between about 10bases and about 25 bases or about 15 bases. In one embodiment, thenuclease domain of the first gene-editing protein and the nucleasedomain of the second gene-editing protein are capable of forming adimer. In another embodiment, the dimer is capable of generating a nickor double-strand break in a target DNA molecule.

Certain embodiments are directed to a therapeutic composition. Otherembodiments are directed to a cosmetic composition. In some embodiments,the composition comprises a repair template. In a further embodiment,the repair template is a single-stranded DNA molecule or adouble-stranded DNA molecule.

Other embodiments are directed to an article of manufacture forsynthesizing a protein or a nucleic acid encoding a protein. In oneembodiment, the article is a nucleic acid. In another embodiment, theprotein comprises a DNA-binding domain. In a further embodiment, thenucleic acid comprises a nucleotide sequence encoding a DNA-bindingdomain. In one embodiment, the protein comprises a nuclease domain. Inanother embodiment, the nucleic acid comprises a nucleotide sequenceencoding a nuclease domain. In one embodiment, the protein comprises aplurality of repeat sequences. In another embodiment, the nucleic acidencodes a plurality of repeat sequences. In a further embodiment, thenuclease domain is selected from: FokI, StsI, StsI-HA, StsI-HA2,StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineeredvariant or biologically active fragment thereof. In one embodiment, thenucleic acid comprises an RNA-polymerase promoter. In anotherembodiment, the RNA-polymerase promoter is a T7 promoter or a SP6promoter. In a further embodiment, the nucleic acid comprises a viralpromoter. In one embodiment, the nucleic acid comprises an untranslatedregion. In another embodiment, the nucleic acid is an invitro-transcription template.

Certain embodiments are directed to a method for inducing a cell toexpress a protein in vivo. Other embodiments are directed to a methodfor altering the DNA sequence of a cell in vivo comprising transfectingthe cell in vivo with a gene-editing protein or inducing the cell toexpress a gene-editing protein in vivo. Still other embodiments aredirected to a method for reducing the expression of a protein ofinterest in a cell in vivo. In one embodiment, the cell is induced toexpress a gene-editing protein, wherein the gene-editing protein iscapable of creating a nick or a double-strand break in a target DNAmolecule. In another embodiment, the nick or double-strand break resultsin inactivation of a gene. Still other embodiments are directed to amethod for generating an inactive, reduced-activity or dominant-negativeform of a protein in vivo. In one embodiment, the protein is survivin.Still other embodiments are directed to a method for repairing one ormore mutations in a cell in vivo. In one embodiment, the cell iscontacted with a repair template. In another embodiment, the repairtemplate is a DNA molecule. In a further embodiment, the repair templatedoes not contain a binding site of the gene-editing protein. In a stillfurther embodiment, the repair template encodes an amino-acid sequencethat is encoded by a DNA sequence that comprises a binding site of thegene-editing protein.

In various embodiments, the repair template is about 20 nucleotides, orabout 30 nucleotides, or about 40 nucleotides, or about 50 nucleotides,or about 60 nucleotides, or about 70 nucleotides, or about 80nucleotides, or about 90 nucleotides, or about 100 nucleotides, or about150 nucleotides, or about 200 nucleotides, or about 300 nucleotides, orabout 400 nucleotides, or about 500 nucleotides, or about 750nucleotides, or about 1000 nucleotides. In various embodiments, therepair template is about 20-1000 nucleotides, or about 20-500nucleotides, or about 20-400 nucleotides, or about 20-200 nucleotides,or about 20-100 nucleotides, or about 80-100 nucleotides, or about50-100 nucleotides.

In various embodiments, the mass ratio of RNA (e.g. synthetic RNAencoding gene-editing protein) to repair template is about 1:10, orabout 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, orabout 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, orabout 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, orabout 8:1, or about 9:1, or about 10:1.

In various embodiments, the molar ratio of RNA (e.g. synthetic RNAencoding gene-editing protein) to repair template is about 1:10, orabout 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, orabout 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, orabout 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, orabout 8:1, or about 9:1, or about 10:1.

In various embodiments, the repair template has a dual function, causinga repair to a gene-edited target sequence and preventing further bindingof a gene-editing protein, thereby reducing or eliminating furthergene-editing (e.g. via the repair template causing a repair that renderswhat was the gene-editing protein binding site no longer suitable forgene-editing protein binding). Accordingly, in some embodiments, thepresent gene-editing methods are tunable to ensure a single gene-editper target site.

Other embodiments are directed to a method for treating a patientcomprising administering to the patient a therapeutically orcosmetically effective amount of a protein or a nucleic acid encoding aprotein. In one embodiment, the treatment results in one or more of thepatient's symptoms being ameliorated. Certain embodiments are directedto a method for treating a patient comprising: a. inducing a cell toexpress a protein of interest by transfecting the cell in vivo with anucleic acid encoding the protein of interest and/or b. reprogrammingthe cell in vivo. In one embodiment, the cell is reprogrammed to a lessdifferentiated state. In another embodiment, the cell is reprogrammed bytransfecting the cell with one or more synthetic RNA molecules encodingone or more reprogramming proteins. In a further embodiment, the cell isdifferentiated. In a still further embodiment, the cell isdifferentiated into one of: a skin cell, a glucose-responsiveinsulin-producing cell, a hematopoietic cell, a cardiac cell, a retinalcell, a renal cell, a neural cell, a stromal cell, a fat cell, a bonecell, a muscle cell, an oocyte, and a sperm cell. Other embodiments aredirected to a method for treating a patient comprising: a. inducing acell to express a gene-editing protein by transfecting the cell in vivowith a nucleic acid encoding a gene-editing protein and/or b.reprogramming the cell in vivo.

Other embodiments are directed to a complexation medium. In oneembodiment, the complexation medium has a pH greater than about 7, orgreater than about 7.2, or greater than about 7.4, or greater than about7.6, or greater than about 7.8, or greater than about 8.0, or greaterthan about 8.2, or greater than about 8.4, or greater than about 8.6, orgreater than about 8.8, or greater than about 9.0. In anotherembodiment, the complexation medium comprises transferrin. In a furtherembodiment, the complexation medium comprises DMEM. In a still furtherembodiment, the complexation medium comprises DMEM/F12. Still otherembodiments are directed to a method for formingnucleic-acid-transfection-reagent complexes. In one embodiment, thetransfection reagent is incubated with a complexation medium. In anotherembodiment, the incubation occurs before a mixing step. In a furtherembodiment, the incubation step is between about 5 seconds and about 5minutes or between about 10 seconds and about 2 minutes or between about15 seconds and about 1 minute or between about 30 seconds and about 45seconds. In one embodiment, the transfection reagent is selected fromTable 1. In another embodiment, the transfection reagent is a lipid orlipidoid. In a further embodiment, the transfection reagent comprises acation. In a still further embodiment, the cation is a multivalentcation. In a still further embodiment, the transfection reagent isN1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(a.k.a. MVL5) or a derivative thereof.

Certain embodiments are directed to a method for inducing a cell toexpress a protein by contacting the cell with a nucleic acid in vivo. Inone embodiment, the cell is a mammalian cell. In another embodiment, thecell is a human cell or a rodent cell. Other embodiments are directed toa cell produced using one or more of the methods of the presentinvention. In one embodiment, the cell is present in a patient. Inanother embodiment, the cell is isolated from a patient. Otherembodiments are directed to a screening library comprising a cellproduced using one or more of the methods of the present invention. Inone embodiment, the screening library is used for at least one of:toxicity screening, including: cardiotoxicity screening, neurotoxicityscreening, and hepatotoxicity screening, efficacy screening,high-throughput screening, high-content screening, and other screening.

Other embodiments are directed to a kit containing a nucleic acid. Inone embodiment, the kit contains a delivery reagent (a.k.a.“transfection reagent”). In another embodiment, the kit is areprogramming kit. In a further embodiment, the kit is a gene-editingkit. Other embodiments are directed to a kit for producing nucleicacids. In one embodiment, the kit contains at least two of:pseudouridine-triphosphate, 5-methyluridine triphosphate,5-methylcytidine triphosphate, 5-hydroxymethylcytidine triphosphate,N4-methylcytidine triphosphate, N4-acetylcytidine triphosphate, and7-deazaguanosine triphosphate or one or more derivatives thereof. Otherembodiments are directed to a therapeutic or cosmetic comprising anucleic acid. In one embodiment, the therapeutic or cosmetic is apharmaceutical composition. In another embodiment, the pharmaceuticalcomposition is formulated. In a further embodiment, the formulationcomprises an aqueous suspension of liposomes. Example liposomecomponents are set forth in Table 1, and are given by way of example,and not by way of limitation. In one embodiment, the liposomes includeone or more polyethylene glycol (PEG) chains. In another embodiment, thePEG is PEG2000. In a further embodiment, the liposomes include1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivativethereof. In one embodiment, the therapeutic comprises one or moreligands. In another embodiment, the therapeutic comprises at least oneof: androgen, CD30 (TNFRSF8), a cell-penetrating peptide, CXCR,estrogen, epidermal growth factor, EGFR, HER2, folate, insulin,insulin-like growth factor-I, interleukin-13, integrin, progesterone,stromal-derived-factor-1, thrombin, vitamin D, and transferrin or abiologically active fragment or variant thereof. Still other embodimentsare directed to a therapeutic or cosmetic comprising a cell generatedusing one or more of the methods of the present invention. In oneembodiment, the therapeutic is administered to a patient for thetreatment of any of the diseases or disorders described herein,including by way of non-limitation, type 1 diabetes, heart disease,including ischemic and dilated cardiomyopathy, macular degeneration,Parkinson's disease, cystic fibrosis, sickle-cell anemia, thalassemia,Fanconi anemia, severe combined immunodeficiency, hereditary sensoryneuropathy, xeroderma pigmentosum, Huntington's disease, musculardystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer,and infectious diseases including: hepatitis and HIV/AIDS.

Other embodiments are directed to a method for reprogramming a cell invivo. In one embodiment, the cell is reprogrammed by contacting the cellwith one or more nucleic acids. In one embodiment, the cell is contactedwith a plurality of nucleic acids encoding at least one of: Oct4protein, Sox2 protein, Klf4 protein, c-Myc protein, Lin28 protein or abiologically active fragment, variant or derivative thereof. In anotherembodiment, the cell is contacted with a plurality of nucleic acidsencoding a plurality of proteins including: Oct4 protein, Sox2 protein,Klf4 protein, and c-Myc protein or one or more biologically activefragments, variants or derivatives thereof. Still other embodiments aredirected to a method for gene editing a cell in vivo. In one embodiment,the cell is gene-edited by contacting the cell with one or more nucleicacids.

Certain embodiments are directed to a method for inducing a cell in vivoto express a protein of interest comprising contacting a cell in vivowith a solution comprising albumin that is treated with an ion-exchangeresin or charcoal and one or more nucleic acid molecules, wherein atleast one of the one or more nucleic acid molecules encodes a protein ofinterest. In one embodiment, the method results in the cell expressingthe protein of interest. In another embodiment, the one or more nucleicacid molecules comprise a synthetic RNA molecule. In one embodiment, thecell is a skin cell. In another embodiment, the cell is a muscle cell.In yet another embodiment, the cell is a dermal fibroblast. In yetanother embodiment, the cell is a myoblast. In one embodiment, theprotein of interest is an extracellular matrix protein. In anotherembodiment, the protein of interest is selected from: elastin, collagen,laminin, fibronectin, vitronectin, lysyl oxidase, elastin bindingprotein, a growth factor, fibroblast growth factor, transforming growthfactor beta, granulocyte colony-stimulating factor, a matrixmetalloproteinase, an actin, fibrillin, microfibril-associatedglycoprotein, a lysyl-oxidase-like protein, and platelet-derived growthfactor. In one embodiment, the solution is delivered to the dermis. Inanother embodiment, the delivering is by injection. In yet anotherembodiment, the delivering is by injection using a microneedle array. Inone embodiment, the solution further comprises a growth factor. Inanother embodiment, the growth factor is selected from: fibroblastgrowth factor and transforming growth factor beta. In yet anotherembodiment, the solution further comprises cholesterol. Otherembodiments are directed a method for inducing a cell in vivo to expressa protein of interest comprising contacting a cell in vivo with asolution comprising cholesterol and one or more nucleic acid molecules,wherein at least one of the one or more nucleic acid molecules encodes aprotein of interest. In one embodiment, the method results in the cellexpressing the protein of interest. Still other embodiments are directedto a method for transfecting a cell in vivo with a nucleic acid moleculecomprising contacting a cell in vivo with a solution comprising albuminthat is treated with an ion-exchange resin or charcoal and a nucleicacid molecule. In one embodiment, the method results in the cell beingtransfected with the nucleic acid molecule. In another embodiment, thenucleic acid molecule is one of: a dsDNA molecule, a ssDNA molecule, adsRNA molecule, a ssRNA molecule, a plasmid, an oligonucleotide, asynthetic RNA molecule, a miRNA molecule, an mRNA molecule, an siRNAmolecule. Still other embodiments are directed to a method for treatinga patient comprising delivering to a patient a composition comprisingalbumin that is treated with an ion-exchange resin or charcoal and oneor more nucleic acid molecules, wherein at least one of the one or morenucleic acid molecules encodes a protein of interest. In one embodiment,the method results in the expression of the protein of interest in thepatient. In another embodiment, the method results in the expression ofthe protein of interest in the dermis of the patient.

Certain embodiments are directed to a cosmetic composition comprisingalbumin that is treated with an ion-exchange resin or charcoal and anucleic acid molecule. Other embodiments are directed to a cosmetictreatment article. In one embodiment, the cosmetic treatment articlecomprises a device configured to deliver a composition to a patient. Inanother embodiment, the nucleic acid molecule encodes elastin protein orcollagen protein. Still other embodiments are directed to a solution fortransfecting a cell in vivo comprising cholesterol or a cholesterolanalog and one or more nucleic acid molecules. In one embodiment, thecholesterol or cholesterol analog is covalently bound to at least one ofthe one or more nucleic acid molecules. In another embodiment, thecholesterol analog is an oxysterol. In yet another embodiment, thecholesterol analog includes one or more of: an A-ring substitution, aB-ring substitution, a D-ring substitution, a side-chain substitution, acholestanoic acid, a cholestenoic acid, a polyunsaturated moiety, adeuterated moiety, a fluorinated moiety, a sulfonated moiety, aphosphorylated moiety, and a fluorescent moiety. In yet anotherembodiment, the method comprises treating the patient with one or moreof: a dermal filler, a neurotoxin (by way of illustration sodium channelinhibitors (e.g., tetrodotoxin), potassium channel inhibitors (e.g.,tetraethylammonium), chloride channel inhibitors (e.g., chlorotoxin andcurare), calcium channel inhibitors (e.g., conotoxin), synaptic vesiclerelease inhibitors (e.g., botulinum toxin and tetanus toxin) and bloodbrain barrier inhibitor (e.g., aluminum and mercury)) and arepair-inducing treatment.

Despite the tendency of transfection reagent nucleic acid complexes toprecipitate, form clumps or otherwise degrade when stored for more thana few minutes, the present inventors have surprisingly discovered thattransfection reagent nucleic acid complexes produced according to someembodiments of the present invention can be frozen and/or can be storedat various temperatures, including room temperature, about 4° C., about−20° C., about −80° C., and about −196° C. for an extended period oftime, for example, for several hours, about 1 day, about 1 week, about 1month, about 1 year, and longer than about 1 year. Some embodiments aretherefore directed to a pharmaceutical formulation comprising syntheticRNA and a transfection reagent, wherein the pharmaceutical formulationis provided in solid form. Other embodiments are directed to apharmaceutical formulation comprising synthetic RNA transfection reagentcomplexes, wherein the synthetic RNA transfection reagent complexes areprovided in solid form. In various embodiments, the synthetic RNAtransfection reagent complexes are provided in frozen form. Variousembodiments are directed to a method for stabilizing nucleic acidtransfection reagent complexes comprising forming nucleic acidtransfection reagent complexes and contacting the nucleic acidtransfection reagent complexes or vessel in which such are containedwith a cryogenic liquid to produce stabilized nucleic acid transfectionreagent complexes. In one embodiment, the nucleic acid transfectionreagent complexes are stabilized for shipment or storage.

Illustrative subjects or patients refers to any vertebrate including,without limitation, humans and other primates (e.g., chimpanzees andother apes and monkey species), farm animals (e.g., cattle, sheep, pigs,goats, and horses), domestic mammals (e.g., dogs and cats), laboratoryanimals (e.g., rodents such as mice, rats, and guinea pigs), and birds(e.g., domestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like). In some embodiments,the subject is a mammal. In some embodiments, the subject is a human.

Definitions

By “molecule” is meant a molecular entity (molecule, ion, complex,etc.).

By “RNA molecule” is meant a molecule that comprises RNA.

By “synthetic RNA molecule” is meant an RNA molecule that is producedoutside of a cell or that is produced inside of a cell usingbioengineering, by way of non-limiting example, an RNA molecule that isproduced in an in vitro-transcription reaction, an RNA molecule that isproduced by direct chemical synthesis or an RNA molecule that isproduced in a genetically-engineered E. coli cell.

By “transfection” is meant contacting a cell with a molecule, whereinthe molecule is internalized by the cell.

By “upon transfection” is meant during or after transfection.

By “transfection reagent” is meant a substance or mixture of substancesthat associates with a molecule and facilitates the delivery of themolecule to and/or internalization of the molecule by a cell, by way ofnon-limiting example, a cationic lipid, a charged polymer or acell-penetrating peptide.

By “reagent-based transfection” is meant transfection using atransfection reagent.

By “medium” is meant a solvent or a solution comprising a solvent and asolute, by way of non-limiting example, Dulbecco's Modified Eagle'sMedium (DMEM), DMEM+10% fetal bovine serum (FBS), saline or water.

By “complexation medium” is meant a medium to which a transfectionreagent and a molecule to be transfected are added and in which thetransfection reagent associates with the molecule to be transfected.

By “transfection medium” is meant a medium that can be used fortransfection, by way of non-limiting example, Dulbecco's ModifiedEagle's Medium (DMEM), DMEM/F12, saline or water.

By “recombinant protein” is meant a protein or peptide that is notproduced in animals or humans. Non-limiting examples include humantransferrin that is produced in bacteria, human fibronectin that isproduced in an in vitro culture of mouse cells, and human serum albuminthat is produced in a rice plant.

By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human Oct4protein (SEQ ID NO: 8), mouse Oct4 protein, Oct1 protein, a proteinencoded by POU5F1 pseudogene 2, a DNA-binding domain of Oct4 protein oran Oct4-GFP fusion protein. In some embodiments the Oct4 proteincomprises an amino acid sequence that has at least 70% identity with SEQID NO: 8, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95%identity with SEQ ID NO: 8. In some embodiments, the Oct4 proteincomprises an amino acid sequence having from 1 to 20 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 8. Or in other embodiments, the Oct4 protein comprises anamino acid sequence having from 1 to 15 or from 1 to 10 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 8.

By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human Sox2protein (SEQ ID NO: 9), mouse Sox2 protein, a DNA-binding domain of Sox2protein or a Sox2-GFP fusion protein. In some embodiments the Sox2protein comprises an amino acid sequence that has at least 70% identitywith SEQ ID NO: 9, or in other embodiments, at least 75%, 80%, 85%, 90%,or 95% identity with SEQ ID NO: 9. In some embodiments, the Sox2 proteincomprises an amino acid sequence having from 1 to 20 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 9. Or in other embodiments, the Sox2 protein comprises anamino acid sequence having from 1 to 15 or from 1 to 10 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 9.

By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human Klf4protein (SEQ ID NO: 10), mouse Klf4 protein, a DNA-binding domain ofKlf4 protein or a Klf4-GFP fusion protein. In some embodiments the Klf4protein comprises an amino acid sequence that has at least 70% identitywith SEQ ID NO: 10, or in other embodiments, at least 75%, 80%, 85%,90%, or 95% identity with SEQ ID NO: 10. In some embodiments, the Klf4protein comprises an amino acid sequence having from 1 to 20 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 10. Or in other embodiments, the Klf4 protein comprises anamino acid sequence having from 1 to 15 or from 1 to 10 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 10.

By “c-Myc protein” is meant a protein that is encoded by the MYC gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human c-Mycprotein (SEQ ID NO: 11), mouse c-Myc protein, I-Myc protein, c-Myc(T58A) protein, a DNA-binding domain of c-Myc protein or a c-Myc-GFPfusion protein. In some embodiments the c-Myc protein comprises an aminoacid sequence that has at least 70% identity with SEQ ID NO: 11, or inother embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQID NO: 11. In some embodiments, the c-Myc protein comprises an aminoacid having from 1 to 20 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 11. Or in otherembodiments, the c-Myc protein comprises an amino acid sequence havingfrom 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 11.

By “erythropoietin” or “erythropoietin protein” is meant a protein thatis encoded by the EPO gene, or a natural or engineered variant,family-member, orthologue, fragment or fusion construct thereof, by wayof non-limiting example, human erythropoietin (SEQ ID NO: 164), mouseerythropoietin, darbepoetin, darbepoetin alfa, NOVEPOETIN, a bindingdomain of erythropoietin or an erythropoietin-GFP fusion protein. Insome embodiments the erythropoietin comprises an amino acid sequencethat has at least 70% identity with SEQ ID NO: 164, or in otherembodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ IDNO: 164. In some embodiments, the erythropoietin comprises an amino acidsequence having from 1 to 20 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 164. Or in otherembodiments, the erythropoietin comprises an amino acid sequence havingfrom 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 164.

By “reprogramming” is meant causing a change in the phenotype of a cell,by way of non-limiting example, causing a n-cell progenitor todifferentiate into a mature β-cell, causing a fibroblast todedifferentiate into a pluripotent stem cell, causing a keratinocyte totransdifferentiate into a cardiac stem cell, causing the telomeres of acell to lengthen or causing the axon of a neuron to grow.

By “reprogramming factor” is meant a molecule that, when a cell iscontacted with the molecule and/or the cell expresses the molecule, can,either alone or in combination with other molecules, causereprogramming, by way of non-limiting example, Oct4 protein, Tertprotein or erythropoietin.

By “germ cell” is meant a sperm cell or an egg cell.

By “pluripotent stem cell” is meant a cell that can differentiate intocells of all three germ layers (endoderm, mesoderm, and ectoderm) invivo.

By “somatic cell” is meant a cell that is not a pluripotent stem cell ora germ cell, by way of non-limiting example, a skin cell.

By “hematopoietic cell” is meant a blood cell or a cell that candifferentiate into a blood cell, by way of non-limiting example, ahematopoietic stem cell or a white blood cell.

By “cardiac cell” is meant a heart cell or a cell that can differentiateinto a heart cell, by way of non-limiting example, a cardiac stem cellor a cardiomyocyte.

By “retinal cell” is meant a cell of the retina or a cell that candifferentiate into a cell of the retina, by way of non-limiting example,a retinal pigmented epithelial cell.

By “skin cell” is meant a cell that is normally found in the skin, byway of non-limiting example, a fibroblast, a keratinocyte, a melanocyte,an adipocyte, a mesenchymal stem cell, an adipose stem cell or a bloodcell.

By “immunosuppressant” is meant a substance that can suppress one ormore aspects of an immune system, and that is not normally present in amammal, by way of non-limiting example, B18R or dexamethasone.

By “single-strand break” is meant a region of single-stranded ordouble-stranded DNA in which one or more of the covalent bonds linkingthe nucleotides has been broken in one of the one or two strands.

By “double-strand break” is meant a region of double-stranded DNA inwhich one or more of the covalent bonds linking the nucleotides has beenbroken in each of the two strands.

By “nucleotide” is meant a nucleotide or a fragment or derivativethereof, by way of non-limiting example, a nucleobase, a nucleoside, anucleotide-triphosphate, etc.

By “nucleoside” is meant a nucleotide or a fragment or derivativethereof, by way of non-limiting example, a nucleobase, a nucleoside, anucleotide-triphosphate, etc.

By “gene editing” is meant altering the DNA sequence of a cell, by wayof non-limiting example, by transfecting the cell with a protein thatcauses a mutation in the DNA of the cell or by transfecting the cellwith a protein that causes a chemical change in the DNA of the cell.

By “gene-editing protein” is meant a protein that can, either alone orin combination with one or more other molecules, alter the DNA sequenceof a cell, by way of non-limiting example, a nuclease, a transcriptionactivator-like effector nuclease (TALEN), a zinc-finger nuclease, ameganuclease, a nickase, a clustered regularly interspaced shortpalindromic repeat (CRISPR)-associated protein, a DNA-repair protein, aDNA-modification protein, a base-modification protein, a DNAmethyltransferase, an protein that causes DNA demethylation, an enzymefor which DNA is a substrate or a natural or engineered variant,family-member, orthologue, domain, fragment or fusion construct thereof.

By “repair template” is meant a nucleic acid containing a region of atleast about 70% homology with a sequence that is within 10 kb of atarget site of a gene-editing protein.

By “repeat sequence” is meant an amino-acid sequence that is present inmore than one copy in a protein, to within at least about 10% homology,by way of non-limiting example, a monomer repeat of a transcriptionactivator-like effector.

By “DNA-binding domain” is meant a region of a molecule that is capableof binding to a DNA molecule, by way of non-limiting example, a proteindomain comprising one or more zinc fingers, a protein domain comprisingone or more transcription activator-like (TAL) effector repeat sequencesor a binding pocket of a small molecule that is capable of binding to aDNA molecule.

By “binding site” is meant a nucleic-acid sequence that is capable ofbeing recognized by a gene-editing protein, DNA-binding protein,DNA-binding domain or a biologically active fragment or variant thereofor a nucleic-acid sequence for which a gene-editing protein, DNA-bindingprotein, DNA-binding domain or a biologically active fragment or variantthereof has high affinity, by way of non-limiting example, an about20-base-pair sequence of DNA in exon 1 of the human BIRC5 gene.

By “target” is meant a nucleic acid that contains a binding site.

Other definitions are set forth in U.S. application Ser. No. 13/465,490,U.S. Provisional Application No. 61/664,494, U.S. ProvisionalApplication No. 61/721,302, International Application No.PCT/US12/67966, U.S. Provisional Application No. 61/785,404, U.S.Provisional Application No. 61/842,874, International Application No.PCT/US13/68118, U.S. Provisional Application No. 61/934,397, U.S.application Ser. No. 14/296,220, U.S. Provisional Application No.62/038,608, U.S. Provisional Application No. 62/069,667, andInternational Application No. PCT/US2015/013949, the contents of whichare hereby incorporated by reference in their entireties.

Selected Sequences SEQ ID NO Description 1 StsI 2 StsI-HA 3 StsI-HA2 4StsI-UHA 5 StsI-UHA2 6 StsI-HF 7 StsI-UHF 8 Oct4 9 Sox2 10 Klf4 11 c-Myc12 BIRC5_exon1 13 BIRC5_exon2 14 BIRC5_exon3 15 BIRC5_exon4 16BIRC5-1.1-L 17 BIRC5-1.1-R 18 BIRC5-1.2-L 19 BIRC5-1.2-R 20 BIRC5-1.3-L21 BIRC5-1.3-R 22 BIRC5-2.1-L 23 BIRC5-2.1-R 24 BIRC5-2.2-L 25BIRC5-2.2-R 26 BIRC5-3.1-L 27 BIRC5-3.1-R 28 CDK1 29 CDK2 30 CDK3 31CDK4 32 CDK5 33 CDK6 34 BIRC5 35 HIF1A 36 RRM2 37 KRAS 38 EGFR 39 MYC 40PKN3 41 KIF11 42 APC 43 BRCA1 44 BRCA2 45 TP53 46 APP 47 HTT 48 IAPP 49MAPT 50 PRNP 51 SNCA 52 SOD1 53 FokI 54 Repeat1 55 Repeat2 56 Repeat3 57EO-GHGG-FokI (“GHGG” disclosed as SEQ ID NO: 547) 58 GHGG-FokI (“GHGG”disclosed as SEQ ID NO: 547) 59 EO-GHGG-StsI (“GHGG” disclosed as SEQ IDNO: 547) 60 GHGG-StsI (“GHGG” disclosed as SEQ ID NO: 547) 61 collagenalpha-1(I) chain preproprotein 62 collagen alpha-2(I) chain precursor 63collagen alpha-1(II) chain isoform 1 precursor 64 collagen alpha-1(II)chain isoform 2 precursor 65 collagen alpha-1(III) chain preproprotein66 collagen alpha-1(IV) chain preproprotein 67 collagen alpha-2(IV)chain preproprotein 68 collagen alpha-3(IV) chain precursor 69 collagenalpha-4(IV) chain precursor 70 collagen alpha-5(IV) chain isoform 1precursor 71 collagen alpha-6(IV) chain isoform A precursor 72 collagenalpha-1(V) chain isoform 1 preproprotein 73 collagen alpha-2(V) chainpreproprotein 74 collagen alpha-3(V) chain preproprotein 75 collagenalpha-1(VI) chain precursor 76 collagen alpha-2(VI) chain isoform 2C2precursor 77 collagen alpha-3(VI) chain isoform 1 precursor 78 collagenalpha-1(VII) chain precursor 79 elastin isoform a precursor 80 elastinisoform b precursor 81 elastin isoform c precursor 82 elastin isoform dprecursor 83 elastin isoform e precursor 84 elastin isoform f precursor85 elastin isoform g precursor 86 elastin isoform h precursor 87 elastinisoform i precursor 88 elastin isoform j precursor 89 elastin isoform kprecursor 90 elastin isoform l precursor 91 elastin isoform m precursor92 protein-lysine 6-oxidase isoform 1 preproprotein 93 protein-lysine6-oxidase isoform 2 94 telomerase reverse transcriptase isoform 1 95telomerase reverse transcriptase isoform 2 96 fibronectin isoform 1preproprotein 97 fibronectin isoform 3 preproprotein 98 fibronectinisoform 4 preproprotein 99 fibronectin isoform 5 preproprotein 100fibronectin isoform 6 preproprotein 101 fibronectin isoform 7preproprotein 102 vitronectin precursor 103 nidogen-1 precursor 104laminin subunit alpha-1 precursor 105 insulin-like growth factor lisoform 1 preproprotein 106 fibroblast growth factor 1 isoform 1precursor 107 fibroblast growth factor 2 108 transforming growth factorbeta-1 precursor 109 transforming growth factor beta-2 isoform 1precursor 110 transforming growth factor beta-2 isoform 2 precursor 111actin, alpha skeletal muscle 112 actin, aortic smooth muscle 113 actin,cytoplasmic 1 114 actin, alpha cardiac muscle 1 proprotein 115 actin,cytoplasmic 2 116 actin, gamma-enteric smooth muscle isoform 1 precursor117 actin, gamma-enteric smooth muscle isoform 2 precursor 118granulocyte colony-stimulating factor isoform a precursor 119granulocyte colony-stimulating factor isoform b precursor 120granulocyte colony-stimulating factor isoform c precursor 121granulocyte colony-stimulating factor isoform d precursor 122platelet-derived growth factor subunit A isoform 1 preproprotein 123platelet-derived growth factor subunit A isoform 2 preproprotein 124platelet-derived growth factor subunit B isoform 1 preproprotein 125platelet-derived growth factor subunit B isoform 2 preproprotein 126platelet-derived growth factor C precursor 127 platelet-derived growthfactor D isoform 1 precursor 128 platelet-derived growth factor Disoform 2 precursor 129 interstitial collagenase isoform 1 preproprotein130 interstitial collagenase isoform 2 131 neutrophil collagenasepreproprotein 132 stromelysin-2 preproprotein 133 macrophagemetalloelastase preproprotein 134 fibrillin-1 precursor 135 fibrillin-2precursor 136 lysyl oxidase homolog 1 preproprotein 137 lysyl oxidasehomolog 2 precursor 138 lysyl oxidase homolog 3 isoform 1 precursor 139lysyl oxidase homolog 3 isoform 2 precursor 140 lysyl oxidase homolog 3isoform 3 141 lysyl oxidase homolog 4 precursor 142microfibrillar-associated protein 2 isoform a precursor 143microfibrillar-associated protein 2 isoform b precursor 144microfibrillar-associated protein 5 precursor 145 disintegrin andmetalloproteinase domain-containing protein 17 preprotein 146desmoglein-2 preproprotein 147 DNA polymerase eta isoform 1 148 DNApolymerase eta isoform 2 149 DNA polymerase eta isoform 3 150ferrochelatase, mitochondrial isoform a precursor 151 ferrochelatase,mitochondrial isoform b precursor 152 filaggrin 153 hyaluronan synthase1 isoform 1 154 hyaluronan synthase 1 isoform 2 155 hyaluronan synthase2 156 hyaluronan synthase 3 isoform a 157 hyaluronan synthase 3 isoformb 158 proopiomelanocortin 159 plakophilin-1 isoform 1a 160 plakophilin-1isoform 1b 161 retinol dehydrogenase 10 162 mitochondrial brown fatuncoupling protein 1 163 tyrosinase precursor 164 erythropoietin 165epoetin alfa 166 darbepoetin alfa 167 NOVEPOETIN 168 NOVECRIT

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 RNA Synthesis

RNA encoding green fluorescent protein (“GFP”), NOVEPOETIN (“EPO”),elastin (“ELN”), tyrosinase (“TYR”), melanocortin-1-receptor (“MC1R”),HAS1, HAS2, HAS3, COL3A1, COL7A1, COL1A1, COL1A2, hTERT, Holly GFP,Fresno RFP, Blitzen Blue, RIBOSLICE gene-editing proteins, TALENs, Cas9,Oct4, Sox2, Klf4, c-Myc-2 (T58A), Lin28, IL2, IL6, IL15, IL22, BMP2,BMP7, BDNF, LIF, BMP6, IL15RA, FGF21, LIF, PTH, KRT5, KRT5-GFP, KRT14,KRT14-GFP, GDF15 and ESM1, and comprising various combinations ofcanonical and non-canonical nucleotides, was synthesized from DNAtemplates using the T7 High Yield RNA Synthesis Kit and the VacciniaCapping System kit with mRNA Cap 2′-O-Methyltransferase (all from NewEngland Biolabs, Inc.), according to the manufacturer's instructions andthe present inventors' previously disclosed inventions (U.S. applicationSer. No. 13/465,490 (now U.S. Pat. No. 8,497,124), InternationalApplication No. PCT/US12/67966, U.S. application Ser. No. 13/931,251,International Application No. PCT/US13/68118, and InternationalApplication No. PCT/US2015/013949, the contents of all of which arehereby incorporated by reference in their entirety) (Table 4). The RNAwas then diluted with nuclease-free water to between 100 ng/μL and 2000ng/μL. For certain experiments, an RNase inhibitor (Superase•In, LifeTechnologies Corporation) was added at a concentration of 1 μL/100 μg ofRNA. RNA solutions were stored at room temperature, 4° C., −20° C. or−80° C. For reprogramming experiments, RNA encoding Oct4, Sox2, Klf4,c-Myc-2 (T58A), and Lin28 was mixed at a molar ratio of 3:1:1:1:1.

TABLE 4 RNA Synthesis Reaction ivT Template Nucleotides Volume/μLYield/μg ELN A, 0.5 7dG, 0.4 5mU, 5mC 20 34.1 ELN A, 0.5 7dG, 0.4 5mU,5mC 20 67.6 GFP A, 0.5 7dG, 0.4 5mU, 5mC 10 60.5 GFP A, 0.5 7dG, 0.45mU, 5hmC 10 25.5 GFP A, G, U, 5hmC 10 58.3 GFP A, 0.5 7dG, U, 5hmC 1047.3 GFP A, 0.5 7dG, 0.4 5mU, 5cC 10 33.8 GFP A, G, U, 5hmC 15 30.3 GFPA, G, U, 5hmC 15 44.6 GFP A, G, U, 5hmC 15 24.7 TYR A, G, U, 5hmC 1545.4 MC1R A, G, U, 5hmC 15 47.5 TYR A, G, U, C 20 67.0 TYR A, G, psU, C20 93.7 TYR A, G, 0.4 5mU, C 20 85.7 TYR A, G, U, 5mC 20 73.4 TYR A, G,U, 5hmC 20 72.7 TYR A, 0.5 7dG, U, C 20 62.7 TYR A, G, psU, 5mC 20 116.3TYR A, G, psU, 5hmC 20 102.4 TYR A, 0.5 7dG, psU, C 20 87.3 TYR A, G,0.4 5mU, 5mC 20 106.5 TYR A, G, 0.4 5mU, 5hmC 20 85.0 TYR A, 0.5 7dG,0.4 5mU, C 20 70.9 TYR A, 0.5 7dG, U, 5mC 20 88.5 TYR A, 0.5 7dG, U,5hmC 20 59.1 TYR A, 0.5 7dG, psU, 5mC 20 7.8 TYR A, 0.5 7dG, psU, 5hmC20 98.0 TYR A, 0.5 7dG, 0.4 5mU, 5mC 20 106.5 TYR A, 0.5 7dG, 0.4 5mU,5hmC 20 82.3 HAS1 A, G, U, 5hmC 20 178.4 HAS2 A, G, U, 5hmC 20 59.3 HAS3A, G, U, 5hmC 20 102.6 TYR A, G, 0.4 5mU, 5hmC 100 377.3 COL3A1 A, G,0.4 5mU, 5hmC 20 108.3 COL7A1 A, G, 0.4 5mU, 5hmC 20 94.6 COL1A1 (20 μL)A, G, 0.4 5mU, 5hmC 20 114.0 COL1A2 (10 μL) A, G, 0.4 5mU, 5hmC 10 31.3TYR A, G, 0.4 5mU, 5hmC 100 249.9 GFP A, G, 0.4 5mU, 5hmC 100 264.0hTERT A, G, 0.4 5mU, 5hmC 100 349.2 GFP A, G, U, 5hC 20 81.7 GFP A, G,U, 0.5 5hC 20 65.4 GFP A, sG, U, C 20 34.7 GFP A, 0.5 sG, U,C 20 47.5GFP A, G, 5hmU, C 20 22.1 GFP A, G, 0.5 5hmU, C 20 28.4 GFP A, G, 5cU, C20 24.4 GFP A, G, 0.5 5cU, C 20 28.4 GFP A, G, 5moU, C 20 39.2 GFP A, G,0.5 5moU, C 20 34.2 GFP A, G, U, C 20 42.0 GFP A, G, 5moU, C 20 53.8 GFPA, G, 5moU, 5hmC 20 101.5 GFP A, G, 0.4 5mU, 0.6 5moU, C 20 98.6 GFP A,G, 0.4 5mU, C 20 99.6 GFP A, G, U, 5mC 20 106.1 GFP A, G, U, C 20 85.7GFP A, G, 5moU, C 100 398.4 hTERT A, G, 5moU, C 20 82.6 COL7A1 A, G,5moU, C 20 34.9 COL7A1 A, G, 5moU, C 100 342.0 Holly GFP A, G, 5moU, C20 36.7 Fresno RFP A, G, 5moU, C 20 72.0 Blitzen Blue A, G, 5moU, C 2030.3 hTERT A, G, 5moU, C 20 49.6 Cas9 A, G, 5moU, C 20 31.6 EPO A, G, U,C 20 101.0 EPO A, G, 5moU, C 20 52.9 EPO A, G, psU, C 20 106.0 COL7A1 A,G, 5moU, C 20 80.2 Oct4 (SEQ ID NO: 8) A, G, 5moU, C 300 1925.5 Sox2(SEQ ID NO: 9) A, G, 5moU, C 100 641.8 Klf4 (SEQ ID NO: 10) A, G, 5moU,C 100 739.0 c-Myc-2 (T58A) A, G, 5moU, C 100 574.0 Lin28 A, G, 5moU, C100 556.0 IL2 A, G, 5moU, C 20 62.4 IL6 A, G, 5moU, C 20 22.2 IL15 A, G,5moU, C 20 50.4 IL22 A, G, 5moU, C 20 63.6 BMP2 A, G, 5moU, C 20 83.2BDNF A, G, 5moU, C 20 45.0 LIF A, G, 5moU, C 20 54.0 BMP6 A, G, 5moU, C20 92.2 IL15RA A, G, 5moU, C 20 91.4 FGF21 A, G, 5moU, C 20 79.2 GFP A,G, 5moU, C 40 181.0 IL2 A, G, 5moU, C 30 99.4 IL6 A, G, 5moU, C 30 31.2IL15 A, G, 5moU, C 30 89.8 IL22 A, G, 5moU, C 30 104.0 BDNF A, G, 5moU,C 30 95.9 BMP2 A, G, 5moU, C 30 112.0 LIF A, G, 5moU, C 30 116.0 PTH A,G, 5moU, C 30 88.4 EPO A, G, 5moU, C 30 83.3 KRT5 A, G, 5moU, C 15 66.6KRT5-GFP A, G, 5moU, C 15 81.1 KRT14 A, G, 5moU, C 15 75.1 KRT14-GFP A,G, 5moU, C 15 90.4 GDF15 A, G, 5moU, C 15 71.1 A1AT TALEN L A, G, 5moU,C 15 56.4 A1AT TALEN R A, G, 5moU, C 15 57.3 A1AT RIBOSLICE L_A A, G,5moU, C 15 74.3 A1AT RIBOSLICE L_B A, G, 5moU, C 15 56.4 A1AT RIBOSLICER_A A, G, 5moU, C 15 60.3 A1AT RIBOSLICE R_B A, G, 5moU, C 15 35.7COL7A1 exon 73 TALEN L A, G, 5moU, C 15 86.48 COL7A1 exon 73 TALEN R A,G, 5moU, C 15 83.66 COL7A1 exon 73 rs3L 50A A, G, 5moU, C 15 103.4COL7A1 exon 73 rs3L 50B A, G, 5moU, C 15 112.8 COL7A1 exon 73 rs3R 50AA, G, 5moU, C 15 81.404 COL7A1 exon 73 rs3R 50B A, G, 5moU, C 15 78.02COL7A1 exon 73 TALEN L A, G, 5moU, C 15 88.924 EA COL7A1 exon 73 TALEN LA, G, 5moU, C 15 75.2 Het COL7A1 exon 73 TALEN R A, G, 5moU, C 15 86.48EA COL7A1 exon 73 TALEN R A, G, 5moU, C 15 62.98 Het COL7A1 exon 73TALEN L A, G, 5moU, C 15 82.7 EA/Het COL7A1 exon 73 TALEN R A, G, 5moU,C 15 69.7 EA/Het HBB exon 1 TALEN L A, G, 5moU, C 15 112.8 HBB exon 1TALEN R A, G, 5moU, C 15 108.1 PD-1 exon 1 TALEN L A, G, 5moU, C 1595.88 PD-1 exon 1 TALEN R A, G, 5moU, C 15 101.52 ESM1 - transcriptvariant 1 A, G, 5moU, C 15 61 ESM1 - transcript variant 2 A, G, 5moU, C15 66

“A” refers to adenosine-5′-triphosphate, “G” refers toguanosine-5′-triphosphate, “U” refers to uridine-5′-triphosphate, “C”refers to cytidine-5′-tri phosphate, “7dG” refers to7-deazaguanosine-5′-triphosphate, “sG” refers tothienoguanosine-5′-triphosphate, “5mC” refers to5-methylcytidine-5′-triphosphate, “5hmC” refers to5-hydroxymethylcytidine-5′-triphosphate, “5cC” refers to5-carboxycytidine-5′-triphosphate, “5fC” refers to5-formylcytidine-5′-triphosphate, “5hC” refers to5-hydroxycytidine-5′-triphosphate, “psU” refers to5-pseudouridine-5′-triphosphate, “5mU” refers to5-methyluridine-5′-triphosphate, “5hmU” refers to5-hydroxymethyluridine-5′-triphosphate, “5cU” refers to5-carboxyuridine-5′-triphosphate, and “5moU” refers to5-methoxyuridine-5′-triphosphate.

Example 2 Preparation of RNA-Transfection-Reagent Complexes

For each microgram of RNA, 1 μg RNA and 1 μL transfection reagent(LIPOFECTAMINE 3000, Life Technologies Corporation) were first dilutedseparately in complexation medium (Opti-MEM, Life TechnologiesCorporation or DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mLsodium selenite+2 μg/mL ethanolamine) to a total volume of between 5 μLand 100 μL each. Diluted RNA and transfection reagent were then mixedand incubated for 10 min at room temperature, according to thetransfection reagent-manufacturer's instructions.

Example 3 Transfection of Cells with Synthetic RNA

Complexes were prepared according to Example 2, and were then addeddirectly to cells in culture. For transfection in 6-well plates, between10 μL and 250 μL of complexes were added to each well of the 6-wellplate, which already contained 2 mL of transfection medium per well.Plates were shaken gently to distribute the complexes throughout thewell. Cells were incubated with complexes for 4 hours to overnight,before replacing the medium with fresh transfection medium (2 mL/well).Alternatively, the medium was not replaced. Volumes were scaled fortransfection in 24-well and 96-well plates.

Example 4 Toxicity of and Protein Translation from Synthetic RNAContaining Non-Canonical Nucleotides

Primary human fibroblasts were transfected according to Example 2, usingRNA synthesized according to Example 1. Cells were fixed and stained20-24 h after transfection using an antibody against Oct4. The relativetoxicity of the RNA was determined by assessing cell density at the timeof fixation.

Example 5 Delivery of Synthetic RNA to the Skin

The complexation reaction shown in Table 5 was prepared using RNAencoding green fluorescent protein (GFP) or collagen, type VII, alphaI(COL7), synthesized according to Example 1. The concentration of the RNAstock solution was 500 μg/mL.

TABLE 5 RNA Complexation Reaction Volume RNA solution tube GFP or COL7RNA  8 μL FactorPlex ™ complexation buffer 42 μL Transfection reagentsolution tube LIPOFECTAMINE 3000 (LIFE TECHNOLOGIES)  4 μL FactorPlex ™complexation buffer 46 μL

Each tube was mixed by pipetting, and the transfection reagent solutiontube was incubated for 30 s at room temperature. The transfectionreagent solution was then transferred to the RNA solution, and thecontents were mixed by rapidly pipetting up and down 10 times. Followinga 10 min incubation, dilutions were prepared according to

TABLE 6 Injection Solutions Complexation FactorPlex ™ RNA Site RNAVolume Volume amount 1 GFP 7.5 μL  22.5 μL   0.3 μg 2 GFP 15 μL 15 μL 0.6 μg 3 GFP 30 μL 0 μL 1.2 μg 4 COL7 30 μL 0 μL 1.2 μg

For each injection, the corresponding solution was drawn into a 3 ccinsulin syringe with an 8 mm, 31 gauge needle (Becton, Dickinson andCompany, Part Number: 328291) and air bubbles were removed. A clearfield was selected on the left forearm of a healthy 33 year-old malehuman subject, and was disinfected with 70% isopropanol and allowed todry. The needle was positioned at an angle of approximately 10° to theanterior (palmar) forearm with bevel facing up, and was inserted untilthe bevel was just covered. 30 μL of the RNA solution was injectedintradermally over the course of approximately 10 sec. A distinct whealappeared during the injection process. The needle was withdrawn, thewheal remained for approximately 1 minute, and no fluid escaped from theinjection site. A total of 4 injections were performed according toTable 6, and all of the injections were performed between 11 and 28minutes following the preparation of the RNA complexation reaction. Noswelling, redness, or soreness occurred as a result of the injections. Asmall amount of bleeding occurred when the needle was removed from sites2 and 4, resulting in the appearance of a small red spot at these sites.

The injection sites were imaged according to the schedule of Table 7,and every 24 hours thereafter for 6 days. Fluorescence images wereacquired using an inverted microscope (Nikon Eclipse TS100) equippedwith an EXFO X-Cite™ 120 fluorescence illumination system and the filtersets shown in Table 7. Fluorescence images were captured using a SonyNEX-7 digital camera (FIGS. 9-12).

TABLE 7 Measurement Parameters Site Time Image Type Exposure Time All  0h Brightfield Automatic 1  0 h FITC 1/10 s 2  0 h FITC 1/10 s 3  0 hFITC 1/10 s 4  0 h FITC 1/10 s 1 12 h FITC 1/10 s 2 12 h FITC 1/10 s 312 h FITC 1/10 s 4 12 h FITC 1/10 s 1 12 h FITC 1/20 s 2 12 h FITC 1/20s 3 12 h FITC 1/20 s 4 12 h FITC 1/20 s All 24 h Brightfield Automatic 124 h FITC 1/20 s 2 24 h FITC 1/20 s 3 24 h FITC 1/20 s 4 24 h FITC 1/20s 1 24 h Cy3.5  ⅕ s 2 24 h Cy3.5  ⅕ s 3 24 h Cy3.5  ⅕ s 4 24 h Cy3.5  ⅕s 1 24 h Cy3  ⅕ s 2 24 h Cy3  ⅕ s 3 24 h Cy3  ⅕ s 4 24 h Cy3  ⅕ s 1 36 hFITC 1/20 s 2 36 h FITC 1/20 s 3 36 h FITC 1/20 s 4 36 h FITC 1/20 s 148 h FITC 1/20 s 2 48 h FITC 1/20 s 3 48 h FITC 1/20 s 4 48 h FITC 1/20s

An independent experiment was carried out using the 1.2 μg dose of GFPRNA, with similar results (FIG. 12).

TABLE 8 Filter Sets Image Type Filter Set Cy3 Chroma SP102V2 Cy3.5Chroma SP103V2 FITC Chroma SP101

Example 6 Transfection of Human Keratinocytes with RNA EncodingNOVEPOETIN

RNA encoding NOVEPOETIN was synthesized according to Example 1 withthree nucleotide combinations: 1) U, G, U, C, 2) U, G, 5moU, C, and 3)U, G, psU, C. Sub-confluent layers of primary human keratinocytescultured in EpiLife medium were transfected in wells of a 6 well plateaccording to Example 3 with 1 μg of RNA per well. 12, 24, 36 and 48 hfollowing transfection, 0.5 mL of medium was removed and 0.5 mL of freshEpiLife medium was added to the plate. After the final medium sampling,the cells were harvested by trypsinization, and total RNA was isolatedusing the RNeasy mini kit (Qiagen). Genomic DNA was digested using DNaseI and RNA was purified. Expression of interferon-β and GAPDH wasmeasured by RT-PCR (FIG. 5).

Example 7 Transfection of Human Cells with RNA Encoding hTERT

RNA encoding human telomerase reverse transcriptase (hTERT) wassynthesized according to Example 1 with the following nucleotides: U, G,5moU, C. A sub-confluent layer of primary human dermal fibroblastscultured in DMEM+10% FBS were transfected in wells of a 24 well plateaccording to Example 3 with 0.25 μg of RNA per well. 12 h aftertransfection, cells were fixed and stained using a 1:50 dilution ofrabbit anti-hTERT antibody (Millipore, Part Number: MABE14) (FIG. 16).

Example 8 High-Efficiency Gene Editing by Repeated Transfection withRIBOSLICE

Primary human fibroblasts were plated in 6-well plates coated withrecombinant human fibronectin and recombinant human vitronectin (eachdiluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, andincubated at room temperature for 1 h) at a density of 10,000 cells/wellin transfection medium. The following day, the cells were transfected asin Example 2 with RNA synthesized according to Example 1. The followingday cells in one of the wells were transfected a second time. Two daysafter the second transfection, the efficiency of gene editing wasmeasured using a mutation-specific nuclease assay.

Example 9 Transfection of Cells with Synthetic RNA ContainingNon-Canonical Nucleotides and DNA Encoding a Repair Template

For transfection in 6-well plates, 1 μg RNA encoding gene-editingproteins targeting exon 16 of the human APP gene, 1 μg single-strandedrepair template DNA containing a PstI restriction site that was notpresent in the target cells, and 6 μL transfection reagent(LIPOFECTAMINE RNAiMAX, Life Technologies Corporation) were firstdiluted separately in complexation medium (Opti-MEM, Life TechnologiesCorporation) to a total volume of 120 μL. Diluted RNA, repair template,and transfection reagent were then mixed and incubated for 15 min atroom temperature, according to the transfection reagent-manufacturer'sinstructions. Complexes were added to cells in culture. Approximately120 μL of complexes were added to each well of a 6-well plate, whichalready contained 2 mL of transfection medium per well. Plates wereshaken gently to distribute the complexes throughout the well. Cellswere incubated with complexes for 4 hours to overnight, before replacingthe medium with fresh transfection medium (2 mL/well). The next day, themedium was changed to DMEM+10% FBS. Two days after transfection, genomicDNA was isolated and purified. A region within the APP gene wasamplified by PCR, and the amplified product was digested with PstI andanalyzed by gel electrophoresis.

Example 10 In Vivo RIBOSLICE Safety Study

40 female NCr nu/nu mice were injected subcutaneously with 5×10⁶MDA-MB-231 tumor cells in 50% Matrigel (BD Biosciences). Cell injectionvolume was 0.2 mL/mouse. The age of the mice at the start of the studywas 8 to 12 weeks. A pair match was conducted, and animals were dividedinto 4 groups of 10 animals each when the tumors reached an average sizeof 100-150 mm³, and treatment was begun. Body weight was measured everyday for the first 5 days, and then biweekly to the end of the study.Treatment consisted of RIBOSLICE BIRC5-1.2 complexed with a vehicle(LIPOFECTAMINE 2000, Life Technologies Corporation). To prepare thedosing solution for each group, 308 μL of complexation buffer (Opti-MEM,Life Technologies Corporation) was pipetted into each of two sterile,RNase-free 1.5 mL tubes. 22 μL of RIBOSLICE BIRC5-1.2 (500 ng/μL) wasadded to one of the two tubes, and the contents of the tube were mixedby pipetting. 22 μL of vehicle was added to the second tube. Thecontents of the second tube were mixed, and then transferred to thefirst tube, and mixed with the contents of the first tube by pipettingto form complexes. Complexes were incubated at room temperature for 10min. During the incubation, syringes were loaded. Animals were injectedeither intravenously or intratumorally with a total dose of 1 μgRNA/animal in 60 μL total volume/animal. A total of 5 treatments weregiven, with injections performed every other day. Doses were notadjusted for body weight. Animals were followed for 17 days. Nosignificant reduction in mean body weight was observed, demonstratingthe in vivo safety of RIBOSLICE gene-editing RNA.

Example 11 Screening of Reagents for Delivery of Nucleic Acids to Cells

Delivery reagents including polyethyleneimine (PEI), various commerciallipid-based transfection reagents, a peptide-based transfection reagent(N-TER, Sigma-Aldrich Co. LLC.), and several lipid-based andsterol-based delivery reagents were screened for transfection efficiencyand toxicity in vitro. Delivery reagents were complexed with RIBOSLICEBIRC5-1.2, and complexes were delivered to HeLa cells in culture.Toxicity was assessed by analyzing cell density 24 h after transfection.Transfection efficiency was assessed by analyzing morphological changes.The tested reagents exhibited a wide range of toxicities andtransfection efficiencies. Reagents containing a higher proportion ofester bonds exhibited lower toxicities than reagents containing a lowerproportion of ester bonds or no ester bonds.

Example 12 High-Concentration Liposomal RIBOSLICE

High-Concentration Liposomal RIBOSLICE was prepared by mixing 1 μg RNAat 500 ng/μL with 3 μL of complexation medium (Opti-MEM, LifeTechnologies Corporation), and 2.5 μL of transfection reagent(LIPOFECTAMINE 2000, Life Technologies Corporation) per μg of RNA with2.5 μL of complexation medium. Diluted RNA and transfection reagent werethen mixed and incubated for 10 min at room temperature to formHigh-Concentration Liposomal RIBOSLICE. Alternatively, a transfectionreagent containing DOSPA or DOSPER is used.

Example 13 In Vivo RIBOSLICE Efficacy Study—Subcutaneous Glioma Model

40 female NCr nu/nu mice were injected subcutaneously with 1×10⁷ U-251tumor cells. Cell injection volume was 0.2 mL/mouse. The age of the miceat the start of the study was 8 to 12 weeks. A pair match was conducted,and animals were divided into 4 groups of 10 animals each when thetumors reached an average size of 35-50 mm³, and treatment was begun.Body weight was measured every day for the first 5 days, and thenbiweekly to the end of the study. Caliper measurements were madebiweekly, and tumor size was calculated. Treatment consisted ofRIBOSLICE BIRC5-2.1 complexed with a vehicle (LIPOFECTAMINE 2000, LifeTechnologies Corporation). To prepare the dosing solution, 294 μL ofcomplexation buffer (Opti-MEM, Life Technologies Corporation) waspipetted into a tube containing 196 μL of RIBOSLICE BIRC5-1.2 (500ng/μL), and the contents of the tube were mixed by pipetting. 245 μL ofcomplexation buffer was pipetted into a tube containing 245 μL ofvehicle. The contents of the second tube were mixed, and thentransferred to the first tube, and mixed with the contents of the firsttube by pipetting to form complexes. Complexes were incubated at roomtemperature for 10 min. During the incubation, syringes were loaded.Animals were injected intratumorally with a total dose of either 2 μg or5 μg RNA/animal in either 20 μL or 50 μL total volume/animal. A total of5 treatments were given, with injections performed every other day.Doses were not adjusted for body weight. Animals were followed for 25days.

Example 14 Liposome Formulation and Nucleic-Acid Encapsulation

Liposomes are prepared using the following formulation: 3.2 mg/mLN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),9.6 mg/mL fully hydrogenated phosphatidylcholine, 3.2 mg/mL cholesterol,2 mg/mL ammonium sulfate, and histidine as a buffer. pH is controlledusing sodium hydroxide and isotonicity is maintained using sucrose. Toform liposomes, lipids are mixed in an organic solvent, dried, hydratedwith agitation, and sized by extrusion through a polycarbonate filterwith a mean pore size of 800 nm. Nucleic acids are encapsulated bycombining 10 μg of the liposome formulation per 1 μg of nucleic acid andincubating at room temperature for 5 minutes.

Example 15 Folate-Targeted Liposome Formulation

Liposomes are prepared using the following formulation: 3.2 mg/mLN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),9.6 mg/mL fully hydrogenated phosphatidylcholine, 3.2 mg/mL cholesterol,2 mg/mL ammonium sulfate, and histidine as a buffer, with 0.27 mg/mL1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (FA-MPEG5000-DSPE) added to the lipid mixture. pH iscontrolled using sodium hydroxide and isotonicity is maintained usingsucrose. To form liposomes, lipids are mixed in an organic solvent,dried, hydrated with agitation, and sized by extrusion through apolycarbonate filter with a mean pore size of 800 nm. Nucleic acids areencapsulated by combining 10 μg of the liposome formulation per 1 μg ofnucleic acid and incubating at room temperature for 5 minutes.

Example 16 Therapy Comprising Liposomal Protein-Encoding RNA

Liposomes encapsulating synthetic RNA encoding a therapeutic protein,synthesized according to Example 1, are prepared according to Example 14or Example 15. The liposomes are administered by injection orintravenous infusion.

Example 17 Generation of Elastin ivT-RNA Template

Total RNA was extracted from neonatal human dermal fibroblasts using theRNeasy mini kit (QIAGEN GmbH), according to the manufacturer'sinstructions. cDNA encoding human elastin was prepared usingMonsterScript™ Reverse Transcriptase (Epicentre Biotechnologies) and theprimer: AAAAAAACCGGT TCATTTTCTCTTCCGGCCAC (SEQ ID NO: 483). An in vitrotranscription (ivT) template was prepared from the cDNA by PCRamplification of the elastin coding sequence (CDS) using the primers: F:AAAAAAGCTAGCATGGCGGGTCTGACG (SEQ ID NO: 484), and R:AAAAAAACCGGTTCATTTTCTCTTCCGGCCAC (SEQ ID NO: 485). The PCR product wasthen purified using agarose gel electrophoresis and the QIAquick GelExtraction Kit (QIAGEN GmbH) and was cloned into a vector containing thehuman beta globin (HBB) 5′ and 3′ untranslated regions and a strongKozak sequence. The vector was amplified, purified, and linearized priorto RNA synthesis.

Example 18 Generation of Tyrosinase ivT-RNA Template

Total RNA was extracted from human epidermal melanocytes using theRNeasy mini kit (QIAGEN GmbH), according to the manufacturer'sinstructions. cDNA encoding human tyrosinase was prepared usingMonsterScript™ Reverse Transcriptase (Epicentre Biotechnologies). An invitro transcription (ivT) template was prepared from the cDNA by PCRamplification of the tyrosinase coding sequence (CDS). The PCR productwas then purified using agarose gel electrophoresis and the QIAquick GelExtraction Kit (QIAGEN GmbH) and was cloned into a vector containing thehuman beta globin (HBB) 5′ and 3′ untranslated regions and a strongKozak sequence. The vector was amplified, purified, and linearized priorto RNA synthesis.

Example 19 Synthesis of Tyrosinase RNA

RNA encoding human tyrosinase was synthesized according to Example 1,using the DNA template of Example 18 and the T7 High Yield RNA SynthesisKit (New England Biolabs, Inc.), according to the manufacturer'sinstructions (Table 4). Samples of the RNA were analyzed by agarose gelelectrophoresis to assess the quality of the RNA. The RNA was thendiluted to 1 μg/μL. The RNA solution was stored at 4C.

Example 20 Increasing Melanin Production in Skin by TransdermalInjection Via Syringe of RNA Encoding Tyrosinase

The RNA of Example 19 was loaded into a syringe and delivered byintradermal injection to the ventral forearm of a healthy 33 year-oldmale patient over the course of approximately 30 seconds.

Example 21 Increasing Melanin Production in Skin by Combined Delivery ofRNA Encoding Tyrosinase and Electroporation

The area of skin treated in Example 20 was exposed to electrical pulsesof between 10V and 155V and between approximately 10 milliseconds andapproximately 1 second using a two-electrode array electricallyconnected to a capacitor. The patient reported a tingling sensation atall voltages and penetration depths. The treated area became darkerafter 24-48 hours. The experiment was repeated several times, withsimilar results.

Example 22 Increasing Melanin Production in Skin by Topical orIntradermal Application of RNA Encoding Tyrosinase

The RNA of Example 19 or the liposomes of Example 16 are applieddirectly to the skin, with or without disruption of the stratum corneumor injected intradermally or delivered by injection to the epidermisusing a dose of one microgram or less per square centimeter. Optionally,an electric field is applied as in Example 21 or using a surface-contactpatch to enhance delivery of the RNA.

Example 23 Increasing Elastin Production in Skin by Transdermal Deliveryof RNA Encoding Elastin

RNA encoding elastin was prepared according to Example 1. The RNA isdelivered as in Example 20, 21, or 22.

Example 24 Increasing Collagen Production in Skin by TransdermalDelivery of RNA Encoding Collagen

RNA encoding collagen was prepared according to Example 1. The RNA isdelivered as in Example 20, 21, or 22.

Example 25 Anemia Therapy Comprising Delivery of RNA Encoding NOVEPOETIN

RNA encoding NOVEPOETIN was prepared according to Example 1. The RNA isdelivered as in Example 20, 21, or 22.

Example 26 Increasing Production of Actin in Skeletal Muscle byIntramuscular Delivery of RNA Encoding Actin

RNA encoding actin is prepared according to Example 1. The RNA isdelivered to the patient via intramuscular injection with or without theuse of an electric field as in Example 20, 21, or 22.

Example 27 Wound Healing Treatment

RNA encoding basic fibroblast growth factor or IL22 is preparedaccording to Example 1. The RNA is delivered as in Example 20, 21, or22.

Example 28 Anti-Scarring Treatment

RNA encoding collagenase is prepared according to Example 1. The RNA isdelivered as in Example 20, 21, or 22.

Example 29 Production of Botulinum Toxin

RNA encoding botulinum toxin is prepared according to Example 1. The RNAis delivered as in Example 20, 21, or 22.

Example 30 Increasing Collagen Production in Skin Cells by Transfectionwith RNA Encoding Collagen I

RNA comprising the coding sequence of the human COL1A1 gene wassynthesized according to Example 1. Primary human dermal fibroblastswere plated in wells of a 24-well plate, and were transfected accordingto Example 2. Between 24 and 72 hours after transfection, the cells werefixed and stained using an antibody targeting collagen I. Manyextracellular deposits of collagen were visible in the transfectedwells.

Example 31 Increasing Collagen Production in Skin Cells by Transfectionwith RNA Encoding Collagen VII

RNA comprising the coding sequence of the human COL7 gene wassynthesized according to Example 1. Primary human dermal fibroblastswere plated in wells of a 24-well plate, and were transfected accordingto Example 2. Between 24 and 72 hours after transfection, the cells werefixed and stained using an antibody targeting collagen VII. Transfectedcells exhibited high levels of collagen VII, compared to anun-transfected control.

Example 32 Increasing Collagen Production in Skin by TransdermalInjection Via Syringe of RNA Encoding Collagen I or Collagen VII

RNA comprising the coding sequence of the human COL1A1 gene or the humanCOL7 gene was synthesized according to Example 1. The RNA is loaded intoa syringe and delivered to the dermis of a patient over the course ofapproximately 30 seconds or as in Example 20, 21, or 22.

Example 33 Increasing Collagen Production in Skin by Combined Deliveryof RNA Encoding Collagen I or Collagen VII and Electroporation

The area of skin treated in Example 32 is exposed to electrical pulsesof between 10V and 155V and between approximately 50 microseconds andapproximately 1 second using a multi-electrode array electricallyconnected to a power source.

Example 34 Storage and Stability of Synthetic RNA Complexes

A complexation reaction using RNA encoding GFP was prepared according toExample 5. Following the 10 min incubation, the complexation reactionwas divided into three equal parts, one of which was diluted 1:10 inFactorPlex™ complexation medium, one of which was diluted 1:10 insterile, nuclease-free water, and one of which was left undiluted. Eachof the three parts was then further divided into four equal parts, oneof which was applied to primary human dermal fibroblasts according toExample 3, one of which was left at room temperature for six hoursbefore applying to primary human dermal fibroblasts according to Example3, one of which was placed at 4° C. for six hours before applying toprimary human dermal fibroblasts according to Example 3, and one ofwhich was snap frozen in liquid nitrogen and placed at −80° C. for sixhours before applying to primary human dermal fibroblasts according toExample 3. The cells were imaged using a fluorescence microscopeapproximately 24 hours after the first transfection (FIG. 17). All wellscontained GFP-positive cells, demonstrating that the synthetic RNAcomplexes were stable and maintained activity in all of the storageconditions tested.

Example 35: In Vivo Analysis of NOVECRIT

RNA encoding NOVEPOETIN was synthesized according to Example 1 with thenucleotide combination A, G, 5moU, C. In this Example, NOVECRIT wasformulated with a lipid delivery vehicle, specifically LIPOFECTAMINE3000. In this Example, NOVECRIT encoded NOVEPOETIN, a novel,high-stability erythropoiesis-stimulating agent.

A 15-day maximum tolerated dose (MTD) study was performed to evaluatesafety (rat, n=62). In vivo toxicology and biodistribution, as well aspharmacodynamics and dose response and therapeutic effect, specificallyon erythropoiesis, were evaluated. Furthermore, the immune response wasmonitored by analysis of cytokines in treated animals.

Sixty-nine, naïve, 8 week old male Sprague-Dawley rats (Rattusnorvegicus) weighing 253 to 274 grams at receipt were used (HARLANLABORATORIES). Animals were acclimated to the study room for at leastseven days prior to dosing. On Day −2 all animals were shaved in thedorsal lumbar area and four intradermal sites (upper left, upper right,lower right and lower left) were designated on the dorsal back using apermanent marker. Sixty-four animals were randomly assigned to fourtreatment groups, with the remaining five animals serving as spares. Twoanimals were dropped from the study because of incomplete dosing.

The following study design was used (total dose volumes (μL) wereconstant):

Dose Dose Level Dose Conc. Vol. Group (μg) Route (μg/mL) (μL) Number ofMales 1 0 Intradermal 0 50 4^(a) + 2^(b) + 2^(h) 2 0.25 Intradermal 5.050 4^(a) + 2^(b) + 2^(c) + 2^(d) + 2^(e) + 2^(f) + 2^(g) + 2^(h) 3 1.0Intradermal 20 50 4^(a) + 2^(b) + 2^(c) + 2^(d) + 2^(e) + 2^(f) +2^(g) + 2^(h) 4 4 × 1.0 μg Intradermal 20 50 4^(a) + 2^(b) + 3^(c) +3^(d) + (4.0 μg 2^(e) + 2^(f) + 2^(g) + 2^(h) total) ID: ^(a)Toxicologyanimals necropsy on Day 15, TK animals with terminal tissue collectionson Days 6^(b) and 24^(c) hours postdose, and on Days 3^(d), 4^(e),6^(f), 8^(g), and 15^(h)

Blood was collected from the vena cava from anesthetized animals priorto necropsy or terminal tissue collection. Whenever possible, blood wascollected via a single draw and then divided appropriately. Bloodspecimens for toxicokinetic analysis were collected from two animals pertime point for Group 1 at 6 hours postdose and on Day 15. For Groups2-4, blood specimens were collected at 6 and 24 hours postdose, and onDays 3, 4, 6, 8, and 15.

Blood specimens for hematology assessment were collected from alltoxicology animals on Day 15 following an overnight fast, and all TKanimals scheduled for terminal tissue collections at 6 and 24 hourspostdose and on Days 8 and 15. Whole blood (1.3 mL) was deposited intoK2EDTA tubes and analyzed using an Advia 120 automated analyzer.

Blood specimens for coagulation assessment were collected from alltoxicology animals on Day 15 following an overnight fast. Coagulationspecimens (1.8 mL) were collected into 3.2% sodium citrate tubes,processed according to standard procedures and analyzed using a STACompact automated analyzer.

Blood specimens for serum chemistry assessments were collected from alltoxicology animals on Day 15 following an overnight fast. Serumchemistry specimens (1 mL) were collected into serum separator tubes,processed according to standard procedures and analyzed using an AU680analyzer.

Blood specimens for cytokine analysis were collected from animalsscheduled for terminal tissue collection at 5 and 24 hours postdose andDay 8. Cytokine specimens (1 mL) were collected into K2EDTA tubes andprocessed to plasma according to standard procedures. TNFα, IL-6, andIFNα cytokine levels in plasma samples were studied. The study measuredthe production of TNFα, IL-6, and IFNα cytokines in plasma samplesfollowing dosing with the test article on Day 1 (6 hours post-dose), Day2 (24 hours post-dose) and Day 8.

Study Outline for Cytokine Analysis Samples:

Dose NOVECRIT Concen- Dose Number Dose Level tration Volume of RatsGroup (μg) (μg/mL) (μL) (Males) Endpoints 1 0 0 50 2b Plasma TNFα, IL-6,and IFNα cytokine analysis 2 0.25 5.0 50 2b + 2c + 2g 3 1.0 20 50 2b +2c + 2g 4 4 × 1.0 μg 20 50 2b + (4.0 μg 2c + 2g total) b= plasma samplescollected 6 hours post-dose, c= plasma samples collected 24 hourspost-dose, g= plasma samples collected 8 days post-dose.

For TNFα analysis, plasma samples were analyzed using acommercially-available assay kit from R&D SYSTEMS (cat. no. RTA00). Therat TNFα ELISA is a solid phase enzyme-linked immunosorbent assay. TheELISA kit employs a TNFα-specific anti-rat monoclonal antibody for solidphase immobilization (pre-coated on the microtiter plate), and HRPconjugated to an anti-rat TNFα polyclonal antibody for detection. Thereference standard provided with the kit is a recombinant rat TNFα. Foreach assay, plasma samples and standards were diluted with the diluentprovided in each respective kit. For the TNFα ELISA, plasma samples werediluted 1:2. The standard curve consisted of six (6) serial 2-foldconcentrations ranging from 800 to 12.5 pg/mL. Controls werereconstituted with diluent, and blanks contained diluent only.

Following sample, standard, control, or diluent addition (50 μL perwell, each in duplicate), plates were incubated for 2 hours at roomtemperature. Following a wash step to remove unbound substances, HRPconjugate was added to each well (100 μL/well), and plates wereincubated for 2 hours at room temperature. Following a second wash step,100 μL/well TMB substrate was added to the plate. The plate wasincubated for 30 minutes at room temperature, protected from light, toallow the color reaction to develop. The reaction was stopped followingthe addition of HCl Stop Solution (100 μL/well). The optical density wasread on a SpectraMax 340 (MOLECULAR DEVICES) plate reader at 450 nm,within 30 minutes following addition of Stop Solution. The intensity ofthe color measured was in proportion to the amount of rat TNFα bound inthe initial step. A standard curve was generated for each assay plate,and test sample TNFα concentrations were determined by interpolation ofabsorbance A450 values from the standard curve and dilution factor. Theassay range for the TNFα kit is 12.5-800 pg/mL, with a minimumdetectable concentration of less than 10 pg/mL (with the 1:2 minimumrequired dilution for plasma samples).

For IL-6 analysis, plasma IL-6 samples were analyzed using acommercially-available assay kit from R&D SYSTEMS (cat. no.R6000B—IL-6). The rat IL-6 ELISA is a solid phase enzyme-linkedimmunosorbent assay. The ELISA kit employs anti-rat IL-6 monoclonalantibody for solid phase immobilization (pre-coated on the microtiterplate), and HRP conjugated to IL-6-specific anti-rat polyclonal antibodyfor detection. The reference standard provided with the kit is arecombinant rat IL-6. Plasma samples were used undiluted (as provided),and standards were diluted with the diluent provided in the kit. Thestandard curve consisted of six (6) serial 2-fold concentrations rangingfrom 4,000 to 62.5 pg/mL. Controls were reconstituted with diluent, andblanks contained diluent only. Following sample, standard, control, ordiluent addition (50 μL per well, each in duplicate), plates wereincubated for 2 hours at room temperature. Following a wash step toremove unbound substances, HRP conjugate was added to each well (100μL/well), and plates were incubated for 2 hours at room temperature.Following a second wash step, 100 μL/well TMB substrate was added to theplate. The plate was incubated for 30 minutes at room temperature,protected from light, to allow the color reaction to develop. Thereaction was stopped following the addition of HCl Stop Solution (100μL/well). The optical density was read on a SpectraMax 340 (MOLECULARDEVICES) plate reader at 450 nm, within 30 minutes following addition ofStop Solution. The intensity of the color measured was in proportion tothe amount of rat IL-6 bound in the initial step. A standard curve wasgenerated for each assay plate, and test sample IL-6 concentrations weredetermined by interpolation of absorbance A450 values from the standardcurve and dilution factor. The assay range for the IL-6 kit is62.5-4,000 pg/mL, with a minimum detectable concentration of less than21 pg/mL.

For IFNα analysis, plasma IFNα samples were analyzed using acommercially-available assay kit from NOVATEINBIO (cat. no.BG-RAT11380). The ELISA is a solid phase enzyme-linked immunosorbentassay. The ELISA kit employs anti-rat IFNα monoclonal antibody for solidphase immobilization (pre-coated on the microtiter plate), and HRPconjugated antibody specific for IFNα for detection. Plasma samples werediluted 1:4, and the standards were used as provided in the kit.Standards consisted of six (6) serial 2-fold concentrations ranging from100 to 3.1 pg/mL. Blanks contained diluent only. Following sample,standard, or diluent addition (50 μL per well, each in duplicate), HRPconjugate was added to each well (100 μL/well), and plates wereincubated for 1 hours at 37° C. Following a wash step, 50 μL/well eachof Chromogen Solution A and Chromogen Solution B were added to theplate. The plate was incubated for 15 minutes at 37° C., protected fromlight, to allow the color reaction to develop. The reaction was stoppedfollowing the addition of Stop Solution (50 μL/well). The opticaldensity was read on a SpectraMax 340 (MOLECULAR DEVICES) plate reader at450 nm. The intensity of the color measured was in proportion to theamount of rat IFNα bound in the initial step. A standard curve wasgenerated for each assay plate, and test IFNα concentrations weredetermined by interpolation of absorbance A450 values from the standardcurve and dilution factor. The assay range for the IFNα kit is 3.1-100pg/mL, with a minimum detectable concentration of less than 1 pg/mL (4pg/mL, with the 1:4 dilution required for plasma samples).

The results of this study were, inter alia, that NOVECRIT was notdetected at the injection site 24 h after dosing (as assayed by RT-PCR).More specifically, NOVECRIT was not detected in any of the followingsamples (RT-PCR): serum (6 h, 24 h, 48 h and 72 h after dosing), liver(6 h and 24 h after dosing), and kidney (6 h and 24 h after dosing).Further, positive controls (spiked with NOVECRIT) yielded a robustsignal in all tissues (as assayed RT-PCR). FIG. 18 depicts singleadministration of NOVECRIT induced a rapid increase and sustained levelof NOVEPOIETIN in serum. The Y axis shows concentration of NOVEPOIETINprotein (mU/mL). This suggests, without wishing to be bound by theory,that the studied RNA therapeutic is able to provide therapeuticallyimportant pharmacodynamic properties. In fact, this PD behavior isvastly improved relative to wild type EPO (which is known in the art tohave a half-life of about 4-12 hours).

Furthermore, serum spiked with NOVECRIT showed near-instant degradationof the RNA (as assayed by RT-PCR). Without wishing to be bound bytheory, this data indicates that the present RNA therapeutics are safeand have little chance of toxicity limitations, for example, liver orkidney toxicities.

FIG. 19 depicts a single administration of NOVECRIT stimulatederythropoiesis, yielding elevated hematocrit for at least 14 days. Theleft panel shows % hematocrit on the Y axis, while the right panel shows% reticulocytes. Accordingly, the studied RNA therapeutic is fullyfunctional.

With respect to cytokines, IL-6 and TNFα cytokine levels were belowassay detection thresholds for all animals at each of the timepointsevaluated in this study. Low levels of IFN-α were only detectable in theDay 8 samples from animals in Groups 3 and 4. FIG. 20 depicts a tablesummarizing TNFα, IL-6, and IFNα cytokine levels in plasma samplescollected from a maximum tolerated dose of NOVECRIT in male SpragueDawley rats. Without wishing to be bound by theory, this data indicatesthat the present RNA therapeutics do not stimulate an unfavorableimmunogenicity which has limited the therapeutic utility of certain RNAtherapeutics.

Example 36: Gene Editing of the COL7A1 Gene

The present RNA-based gene editing approaches were applied to the COL7A1gene. This gene is of interest because, inter alia, it is frequentlyinvolved in dystrophic epidermolysis bullosa. FIG. 21 depicts a SURVEYORassay using the DNA of primary adult human dermal fibroblaststransfected with RNA TALENs targeting the sequence TGAGCAGAAGTGGCTCAGTG(SEQ ID NO: 467) and TGGCTGTACAGCTACACCCC (SEQ ID NO: 468), locatedwithin the COL7A1 gene. The bands present in the +RNA lane indicateediting of a region of the gene that is frequently involved indystrophic epidermolysis bullosa. FIG. 22 depicts another SURVEYOR assayusing the DNA of primary adult human dermal fibroblasts transfected withRNA TALENs, now targeting the sequence TTCCACTCCTGCAGGGCCCC (SEQ ID NO:469) and TCGCCCTTCAGCCCGCGTTC (SEQ ID NO: 470), located within theCOL7A1 gene. The bands present in the +RNA lane indicate editing of aregion of the gene that is frequently involved in dystrophicepidermolysis bullosa. This data points to, among others, a gene editingapproach to the treatment of certain genetic disorders such asdystrophic epidermolysis bullosa.

Example 37: Gene-Editing of the MYC Gene Using a Synthetic RNA withNon-Canonical Nucleotides

Experiments were conducted with in vitro transcribed synthetic RNAmolecules containing non-canonical nucleotides and encoding gene-editingproteins. The immunogenicity and the gene-editing efficiency of in vitrotranscribed synthetic RNA molecules having (1) only pseudouridine (psU)as a non-canonical nucleotide; (2) only 5-methylcytidine (5mC) as anon-canonical nucleotide; and (3) both of pseudouridine and5-methylcytidine as non-canonical nucleotides was evaluated (as well ascontrols).

Specifically, RNA containing the following nucleotide combinations: (i)A,G,U,C, (ii) A,G,psU,C, (iii) A,G,U,5mC, and (iv) A,G,psU,5mC, andencoding TALEN pairs targeting the following DNA sequences, which can befound within the MYC gene: TCGGCCGCCGCCAAGCTCGT (SEQ ID NO: 474) andTGCGCGCAGCCTGGTAGGAG (SEQ ID NO: 475), were synthesized according to themethods described herein. Human dermal fibroblasts (MA001SK) were platedin 6-well and 24-well tissue culture plates in DMEM with 10% FBS at100,000 and 10,000 cells per well, respectively. The next day, the cellswere transfected in the 6-well plate with 2 μg of RNA (1 μg for eachcomponent of the TALEN pair) and the cells were transfected in the24-well plate with 0.2 μg of RNA (0.1 μg for each component of the TALENpair) according to the methods described herein. 24 hours aftertransfection, the total RNA from the cells in the 24-well culture platewas isolated using an RNeasy Mini Kit (74106; QIAGEN), includingisolating the total RNA from a sample of cells that had not beentransfected with RNA (negative control; “Neg.” in FIG. 23). The genomicDNA was removed by a 15 minute digestion with DNase I (RNase-Free)(M0303L; NEW ENGLAND BIOLABS) and the reaction purified using an RNeasyMini Kit. 1 μL of total RNA was used to assess gene expression byreal-time RT-PCR using TAQMAN gene-expression assays (APPLIEDBIOSYSTEMS) designed to detect expression of the immunogenicity markersTLR3, IFIT1, and IFIT2 (FIG. 23). The data were normalized to both thepositive experimental control sample (“A,G,U,C”) and to a loadingcontrol (GAPDH).

48 hours after transfection, the genomic DNA was isolated from the cellsin the 6-well culture plate using a DNeasy Blood and Tissue Kit (69506;QIAGEN), including from a sample of cells that had not been transfectedwith RNA (negative control, “Neg.” in FIG. 24). A 970 bp region of theMYC gene surrounding the predicted TALEN cut location was amplifiedusing a 35 cycle 2-step PCR reaction containing the following primers:TAACTCAAGACTGCCTCCCGCTTT (SEQ ID NO: 476) and AGCCCAAGGTTTCAGAGGTGATGA(SEQ ID NO: 477). 160 ng was hybridized in 5 μL of amplified sequencefrom RNA-treated cells to 160 ng in 5 μL of amplified sequences fromuntreated MA001SK cells by mixing the two sequences with 0.5 μL of 1MKCl and 0.5 μL of 25 mM MgCl₂ and running the following program in athermocycler: 95° C. for 10 minutes; 95° C. to 85° C. at 0.625 C/s; 85°C. to 25° C. at 0.125 C/s. The SURVEYOR assay was performed by adding0.5 μL of SURVEYOR nuclease and 0.5 μL of Enhancer from the SURVEYORMutation Detection Kit (7060201; INTEGRATED DNA TECHNOLOGIES) to thehybridized product, mixing, and incubating at 42° C. for 25 minutes. Theprotocol above was also used to process the positive control DNA sampleprovided with the SURVEYOR Mutation Detection Kit as a positiveexperimental control for the SURVEYOR Assay (“Assay Pos.” in FIG. 24).Samples were analyzed by agarose gel electrophoresis (FIG. 24). For eachsample, gene-editing efficiency was calculated as a ratio of theintensity of the digested bands (indicated by “*” in FIG. 24) to that ofthe undigested band.

As shown in FIG. 23 below, the samples from cells transfected with thepositive control RNA (A,G,U,C), and the samples from cells transfectedwith RNA containing either pseudouridine or 5-methylcytidine exhibitedupregulation of all three of the immunogenicity markers TLR3, IFIT1, andIFIT2. The sample from cells transfected with RNA containing bothpseudouridine and 5-methylcytidine exhibited negligible upregulation ofthe immunogenicity markers (less than 0.01-fold of the positivecontrol), demonstrating that in vitro transcribed synthetic RNA withboth pseudouridine and 5-methylcytidine and encoding a gene-editingprotein can evade detection by the innate-immune system of mammaliancells.

Further, as shown in FIG. 24 below, the sample from cells transfectedwith RNA containing both pseudouridine and 5-methylcytidine exhibitedhighly efficient gene editing (41.7%), which was greater than theefficiency exhibited by samples from cells transfected with RNAcontaining pseudouridine alone (35.2%), demonstrating that in vitrotranscribed synthetic RNA comprising both pseudouridine and5-methylcytidine and encoding a gene-editing protein can both (i)gene-edit mammalian cells at high efficiency, and (ii) gene-editmammalian cells at higher efficiency than in vitro transcribed syntheticRNA comprising pseudouridine and not comprising 5-methylcytidine.

Example 38: COL7A1 Gene Editing and Repair in Human Cells

RNA encoding gene editing proteins targeting the following sequences inthe COL7A1 gene was synthesized according to Example 1:TGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 473) and TGGCTGTACAGCTACACCCC (SEQ IDNO: 468) (see also table below).

RNA Synthesis

Reaction ivT Template Nucleotides Volume/μL Yield/μg COL7A1 TALEN 1L A,G, 5moU, C 20 120.528 COL7A1 TALEN 1R A, G, 5moU, C 20 120.204 COL7A1TALEN 1L A, G, 5moU, C 15 81.94 COL7A1 TALEN 1R A, G, 5moU, C 15 61.88

50,000 primary human epidermal keratinocytes (HEKn, Gibco) were platedin wells of 6-well plates in EpiLife+Supplement S7. The next day, cellswere transfected according to Example 3 with 1 μg of RNA encoding eachcomponent of the gene editing pair and 2 μg of a single-stranded DNArepair template having a length of 60, 70, 80, 90 or 100 nucleotides(“nt”). 48 hours after transfection, genomic DNA was purified. A segmentof the COL7A1 gene was amplified using the primers GCATCTGCCCTGCGGGAGATC(SEQ ID NO: 478) and CCACGTTCTCCTTTCTCTCCCCGTTC (SEQ ID NO: 479), whichproduce a 535 bp amplicon. The efficiency of gene editing was assessedusing T7 Endonuclease I (“T7E1”, New England Biolabs) according to themanufacturer's instructions. Bands of approximately 385 bp and 150 bpindicate successful gene editing. FIG. 25 and FIG. 29 show the result ofdigestion with T7EI, analyzed by agarose gel electrophoresis. FIG. 27and FIG. 29 show the result of digestion with MluI-HF, analyzed byagarose gel electrophoresis. Because the repair template contains thesequence ACGCGT (SEQ ID NO: 480), digestion of the amplified productwith MluI-HF (New England Biolabs) produces bands of approximately 385bp and 150 bp in the case of successful gene repair.

RNA encoding gene editing proteins targeting the following sequences inthe COL7A1 gene was synthesized according to Example 1:TGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 473) and TGGCTGTACAGCTACACCCC (SEQ IDNO: 468). 50,000 primary human epidermal keratinocytes (HEKn, Gibco)were plated in wells of 6-well plates in Epi Life+Supplement S7. Thenext day, cells were transfected according to Example 3 with 1 μg of RNAencoding each component of the gene editing pair and 1-4 μg of an 80nucleotide single-stranded DNA repair template. 48 hours aftertransfection, genomic DNA was purified. A segment of the COL7A1 gene wasamplified using the primers GCATCTGCCCTGCGGGAGATC (SEQ ID NO: 481) andCCACGTTCTCCTTTCTCTCCCCGTTC (SEQ ID NO: 482), which produce a 535 bpamplicon. The efficiency of gene editing was assessed using T7Endonuclease I (“T7E1”, New England Biolabs) according to themanufacturer's instructions. Bands of approximately 385 bp and 150 bpindicate successful gene editing. FIG. 26 show the result of digestionwith T7EI, analyzed by agarose gel electrophoresis. FIG. 28 show theresult of digestion with MluI-HF, analyzed by agarose gelelectrophoresis. Because the repair template contains the sequenceACGCGT (SEQ ID NO: 480), digestion of the amplified product with MluI-HF(New England Biolabs) produces bands of approximately 385 bp and 150 bpin the case of successful gene repair.

Example 39: Expression of BMP7 Variants in Human Cells

RNA encoding wild type BMP7 and RNA encoding variants of BMP7 wassynthesized according to Example 1 (see also table below).

RNA Synthesis

Reaction ivT Template Nucleotides Volume/μL Yield/μg BMP7 Wild Type A,G, 5moU, C 15 125.8 BMP7 Variant A A, G, 5moU, C 15 120.36 BMP7 VariantB A, G, 5moU, C 15 143.14 BMP7 Variant C A, G, 5moU, C 15 106.42

50,000 primary human dermal fibroblasts (MA001SK, Factor Bioscience) or100,000 primary human epidermal keratinocytes (HEKn, Gibco) were platedin DMEM+10% FBS or EpiLife+Supplement S7, respectively. Cells weretransfected according to Example 3 with 1 μg of RNA encoding wild typeBMP7 or a variant thereof. 24 hours after transfection, the medium wassampled and secreted BMP7 levels were measured with a human BMP7 ELISAkit (ab99985, Abcam) using medium diluted 10-fold according to themanufacturer's instructions. Secreted BMP7 levels were determined bymeasuring 450 nm absorbance using a microplate reader (EMax Plus,Molecular Devices). Secreted BMP7 levels are shown in FIG. 30.

Example 40: Expression of Parathyroid Hormone (PTH) in Human Cells

RNA encoding PTH was synthesized according to Example 1 (see also tablebelow).

RNA Synthesis

Reaction ivT Template Nucleotides Volume/μL Yield/μg PTH A, G, 5moU, C15 51.68

100,000 human epidermal keratinocytes (HEKn, Gibco) were plated inEpiLife+Supplement S7. Cells were transfected according to Example 3with 1 μg of RNA encoding PTH. 24 hours after transfection, the mediumwas sampled and secreted PTH levels were measured using a human PTHELISA kit (EIA-PTH-1, Ray Biotech) according the manufacturer'sinstructions. Secreted PTH levels were determined by measuring 450 nmabsorbance using a microplate reader (EMax Plus, Molecular Devices).Secreted PTH levels are shown in FIG. 31.

Example 41: Intradermal, Subcutaneous, Rectal and Nasal Administrationof NOVECRIT for the Treatment of Anemia

A repeat dose toxicity study of Novecrit was conducted for the treatmentof anemia. Specifically, 8-10 weeks old male Sprague Dawley rats wereadministered with Novecrit via intradermal, subcutaneous, rectal, ornasal routes once per day on days 1, 8, and 15. The animals wereassigned to groups and treated as indicated in the table below:

Dose Dose Number of Group Test Concentration Volume Animals Group ColorArticle Dose Route Dose Level (μg) (μg/mL) (μL) Males 1 White ControlIntradermal 0.0 0.0 50 3^(a) + 8^(b) 2 Yellow Novecrit Intradermal 4X5.0 50 3^(a) + 8^(b) (0.25 μg/ injection; 1.0 μg total) 3 Green NovecritSubcutaneous 4.0 20.0 200 3^(a) + 8^(b) 4 Blue Novecrit Rectal 4.0 20.0200 3^(a) + 8^(b) 5 Red Novecrit Nasal 4.0 20.0 200 3^(a) + 8^(b) Note:Total dose volume (μL/per animal) are constant ^(a)Toxicity animals(also called main study animals), necropsy on Day 44 ^(b)TK animals,euthanized as n = 2/time point on Days 2, 16, 23 and 44

More particularly, Group 1 was dosed via intradermal injection. Eachdose was administered in four intradermal injections of 50 μL/injectionfor a total of 200 uL per animal. Injections were carried out onpreviously marked sites near the midline of the dorsal lumbar area(upper left, upper right, lower left and lower right quadrants). Group 2was dosed four intradermal injections at 0.25 μg each (1.0 μg total)into previously marked sites near the midline of the dorsal lumbar area(upper left, upper right, lower left and lower right quadrants). Group 3was dosed by subcutaneous injections into an area of the back located inthe lower dorsal thoracic/lumbar region. Group 4 was dosed by rectaladministration. The animal was manually restrained and the abdomen ofeach rat was manually palpated to remove any fecal matter. If deemednecessary, the rectum was lavaged with up to 2 ml of saline for enemafollowed by manual palpation of the abdomen to remove the fecal matter,if any. Novecrit was drawn into a syringe and an appropriately sizedgavage needle with a rounded tip (ball) was attached to the syringe. Alubricant jelly was applied to the insertion device to aid withinsertion; it was advanced approximately 1 cm into the lumen of thecolon and the Novecrit instilled. The rat was then maintained in ahead-down position for approximately 20-30 seconds to limit theexpulsion of solution. Group 5 was dosed via nasal route. The animal wasanesthetized (per SNBL USA SOP). The animal was laid on its back withthe head elevated. The dose was dispensed slowly into the nares.Approximately half the dose volume was administered to one nare. Theremaining dose volume was administered to another nare. The first dosewas administered on Day 1, and the last dose on Day 15.

The rats were clinically monitored including their food consumption andbody weight. In addition, blood was collected for hematology,coagulation, and serum chemistry analysis as indicated below:

Toxicity Group Specimen collection frequency Time Point HematologyCoagulation Serum Chemistry Day 44 1X 1X 1X (n = 3/Group) (n = 3/Group(n = 3/Group) X = Number of times procedure performed

Analysis was done on the toxicokinetic group as follows:

In-text Table 3: Toxicokinetic Group Specimen collection frequency TimePoint Hematology^(a) TK Cytokines Acclimation 1X — — (n = 8/Group) Day 2— 1X 1X (n = 2/Group, (n = 2/Group, 24 hours postdose) 24 hourspostdose) Day 8 Pre-dose — — (n = 6/Group) Day 15 Pre-dose — — (n =6/Group) Day 16 — 1X 1X (n = 2/Group, (n = 2/Group, 24 hours postdose)24 hours postdose) Day 22 1X — — (n = 4/Group) Day 23 — 1X 1X (n =2/Group) (n = 2/Group) Day 29 1X — — (n = 2/Group) Day 36 1X — — (n =2/Group) Day 43 1X — — (n = 2/Group) Day 44 — 1X 1X (n = 2/Group) (n =2/Group) X = Number of times procedure performed TK = toxicokinetics

Terminal necropsy for toxicity animals occurred on Day 44. TK animalswere euthanized on Days 2, 16, 23, and 44. Pathological analysis wasconducted on the animals.

As shown in FIG. 32, administration of NOVECRIT stimulatederythropoiesis resulting in elevated hematocrit for at least 14 days inall four study groups compared to control.

Example 42: Intradermal Administration of RNA Encoding BMP7 Variants forthe Treatment of Diabetic Nephropathy

A ZDSD rat model was utilized to study the effects of RNAs encoding BMP7variants for the prevention and treatment of diabetic nephropathy.Specifically, ZDSD rats were treated with RNAs encoding BMP7 variantsadministered intradermally. A schematic of the study design is providedin FIG. 33. The animals were assigned to groups and treated as indicatedin the table below:

Dose Dose Dose Route Group No. of Level Conc. Volume of Number TreatmentAnimals (μg) (μg/ml) (ml/kg) Frequency Admin. 1 Vehicle 24 0 0 0.1 2x/wkI.D. 2 FTB-F1 (wt 6 1 5 0.1 2x/wk I.D. BMP7) 3 FTB-F2 6 1 5 0.1 2x/wkI.D. (BMP7 variant A) 4 FTB-F3 6 1 5 0.1 2x/wk I.D. (BMP7 variant B) 5Lisinopril 6 Standard Standard 5   Continuous Diet 6^(a) Vehicle 20 0 00.2/rat 3x/wk I.D. 7^(a) FTB-F2 10 2 10  0.2/rat 3x/wk I.D. (BMP7variant A) ^(a)Group 1 animals at the end of the “Prevention” arm wererandomized into Groups 6 and 7 (n = 20 or 10 each, respectively) todefine the “Treatment” arm. The remaining 6 animals from Group 1 wereeuthanized for kidney collection and blood draw at the end of the“Prevention” arm.

To study the effect of the RNAs on prevention of diabetic nephropathy,animals were delivered from the barrier (PCO-5638) to the vivarium(PCO-Rm C) and allowed 3 days for acclimation. All animals were placedon 5SCA diabetogenic diet for 3 weeks (weekly body weight measurementswere taken). Animals were then returned to regular 5008 diet for theduration of the study (weekly body weight measurements were taken).After 2 weeks on 5008 diet, body weight measurement, blood draw (500 ul)and a 24 hour baseline urine collection (urinary albumin and creatininemeasurements) were performed on all animals. 36 rats were randomized togroups 1, 2 and 3 based on body weight, glucose and urinary albumin.Groups 1, 2, 3 and 4 received either vehicle or test article (i.d.) viaintradermal administration (2×/week, Mon and Thur). Group 5 wasadministered Lisinopril admixed in the 5008 diet (food consumption willbegin on this group of animals). Blood draws (500 ul, tail vein) weretaken from Groups 2, 3 and 4, 24 hours after each dose for the first 2weeks and then 1×/week thereafter. After 4 weeks of dosing, blood samplevia tail vein (500 ul) was taken from all animals (Groups 1-5). At theend of 8 weeks of dosing, a 24 hour urine collection was performed(urinary albumin and creatinine measurements) together with a tail vein(500 ul) blood sample from Group 1 (n=18 going into the Treatment arm).Results for urine volume, creatinine, and albumin is shown in FIG. 35.As shown in FIG. 35, treatment with RNA encoding BMP7 Variant A resultedin reduced levels of urine albumin in rats afflicted with diabeticnephropathy compared to the control, indicating superior kidney functionin the treated animals compared to the control animals (placebo group).To terminate the study, all animals were euthanized with CO₂asphyxiation and a blood draw was performed via cardiac puncture tocollect serum for BUN, creatinine and glucose, and plasma for compoundexposure and biomarker analysis. Both kidneys were dissected and fixedfor histology. FIG. 34 shows the serum level of BMP7 protein in Groups1, 2, 3, and 4. Results indicate that the serum levels of BMP7 proteinin these groups were as follows: Group 4, Group 3>Group 2, Group 1.

A study was also conducted to analyze the effect of the RNAs encodingBMP7 variants on treating diabetic nephropathy. Specifically, thevehicle animals from Group 1 (Prevention arm, n=18) were randomizedbased on body weight, glucose and urine albumin (Groups 6, n=20; 7,n=10;). Animals received either Vehicle or BMP7 variant-encoding RNAs(intradermal, 2×/week, Mon and Thur), groups 6 and 7, respectively.After 4 weeks of treatment, animals were euthanized with CO₂asphyxiation and a blood draw will be performed via cardiac puncture tocollect serum for BUN, creatinine and glucose, and plasma for compoundexposure and biomarker analysis. Both kidneys were dissected and fixedfor histology. As shown in FIG. 36, treatment with RNA encoding BMP7Variant A significantly decreased urine albumin level in rats afflictedwith diabetic nephropathy (p=0.0015), indicating superior kidneyfunction in the treated animals. In addition, the treatment group showedsignificantly lower kidney weight at termination (p=0.018), andsignificantly lower serum creatinine at termination (p=0.016). Theseresults suggested that BMP7 variant-encoding RNA effectively treateddiabetic nephropathy and promoted superior kidney function compared tothe control animals (placebo group).

An illustrative study protocol is provided below:

Week Day Procedure 15 −7 Animals arrive in Room C from the barrier weeksold 0 BW*, Stat Strip, and Place animals on 5SCA for 3 wks  1 7 BW*,Stat Strip  2 14 BW*, Stat Strip  3 21 BW*, Stat Strip, put animals on5008  4 28 BW*, Stat Strip 34 BW*, Tail vein blood draw and randomizefor Prevention arm (Grps 1, 2, 3, 4, 5)  5 35 Begin 24 hr urinecollection 36 Collect 24 hr urine 39 BW* Intradermal dosing Grps 1, 2, 3and 4, and put Grp 5 on admixed 5008 diet Begin food consumption ongroup 5 40 Collect 24 blood sample for exposure (gps 2, 3, & 4) 42Dosing 43 Collect 24 blood sample for exposure (gps 2, 3, & 4)  6 (1 P)46 BW*, Dosing Food consumption group 5 47 Collect 24 blood sample forexposure (gps 2, 3, & 4) 49 Dosing 50 Collect 24 blood sample forexposure (gps 2, 3, & 4)  7 (2 P) 53 BW*, Dosing Food consumption group5 54 Collect 24 blood sample for exposure (gps 2, 3, & 4) 56 Dosing  8(3 P) 60 BW*, Dosing Food consumption group 5 61 Collect 24 blood samplefor exposure (gps 2, 3, & 4) 63 Dosing  9 (4 P) 67 BW*, Dosing Foodconsumption group 5 Blood draw (tail vein) 68 Blood draw (all groups)Collect 24 blood sample for exposure (gps 2, 3, & 4) 70 Dosing 10 (5 P)74 BW*, Dosing Food consumption group 5 75 Collect 24 blood sample forexposure (gps 2, 3, & 4) 77 Dosing 11 (6 P) 81 BW*, Dosing Foodconsumption group 5 Collect 24 blood sample for exposure (gps 2, 3, & 4)84 Dosing 12 (7 P) 88 BW*, Dosing Food consumption group 5 89 Collect 24blood sample for exposure (gps 2, 3, & 4) 91 Dosing 13 (8 P) 95 BW*,dosing Food consumption group 5 Blood draw (tail vein) (all groups) 96Begin 24 hr urine collection 97 Collect 24 hr urine Terminate Groups 1(n = 6), 2, 3, 4 and 5 cardiac puncture blood draw, and collect kidneys,weigh and fix. 98 BW* and randomize (BW*, glucose, albumin) Group 1 (n =18) rats into Groups 6, 7 and 8 Dose Food Consumption group 8 103 Dose14 (1 T) 106 BW*, Dosing Food Consumption group 8 110 Dosing 15 (2 T)113 BW*, Dosing Food Consumption group 8 117 Dosing 16 (3 T) 120 BW*,Dosing Food Consumption group 8 124 Dosing 17 (4T) 127 BW*, Terminate,cardiac puncture blood draw, and collect kidneys, weigh and fix. *BW:body weight measurement

In an illustrative study, the protocol was amended from above towardsthe end of the study as follows:

Week [Treatment BW Arm] Day Begin 24 hr urine collection 81 Collect 24hr urine BW, Blood draw Randomize (BW, glucose, albumin) rats intoGroups 6 and 7 1 T BW, Dose 12 hr PK sample Dose Dose and Begin 24 hrUrine collection 89 Collect 24 hr Urine and Dose 2 T BW, Dose 12 hr PKsample Dose Begin 24 hr Urine collection 96 Collect 24 hr Urine and Dose3 T BW, Dose 12 hr PK sample Dose Begin 24 hr Urine collection Collect24 hr Urine and Dose (last dose) 4 T BW No activity Begin 24 hr urinecollection Collect 24 hr urine BW, Terminate, cardiac puncture blooddraw, and collect kidneys, weigh and fix.

During both studies, pathological analysis is conducted as follows:

Specimen Anti- Specimen Clinical to be Method of Coag- to be ChemistryMethod of Collected Collection ulant Analyzed Analyte Analysis Urine 24hr NA Urine Creatinine/ AU480/ Metabolism Albumin ICL Elisa Cages WholeTail Vein None Serum BUN AU480/ Blood Cardiac Creatinine SponsorPuncture Glucose/ BUN Creatinine Whole (Grps 1, 2, None Serum Glucose/AU480/ Blood 3, 4 and 5; Cmpd Exp & Sponsor and 6, 7, 8) Biomarker

Altogether, these results suggested that RNAs encoding BMP-7 variantseffectively prevented the development of diabetic nephropathy in rats.In addition, these RNAs also effectively treated and restored kidneyfunctions in rats already afflicted with diabetic nephropathy. RNAencoding BMP7 Variant A was particularly effective at preventing andtreating diabetic nephropathy in rats.

Example 43: Transfection of Human Keratinocytes with RNA Encoding BDNF,BMP-2, BMP-6, IL-2, IL-6, IL-15, IL-22, LIF or FGF-21

100,000 human epidermal keratinocytes (HEK, Gibco) were plated inEpiLife+Supplement S7. Cells were transfected according to Example 3with 2 μg of RNA encoding BDNF, BMP-2, BMP-6, IL-2, IL-6, IL-15, IL-22,LIF or FGF-21. 24 hours after transfection, the medium was sampled andsecreted protein levels were measured using a human ELISA kit (see Tablebelow) according the manufacturer's instructions. Secreted proteinlevels were determined by measuring 450 nm absorbance using a microplatereader (EMax Plus, Molecular Devices). Secreted protein levels are shownin FIG. 34, panels A-I.

Protein Part Number Vendor BDNF DBNT00 R&D Systems BMP-2 DBP200 R&DSystems BMP-6 ab99984 Abcam IL-2 D2050 R&D Systems IL-6 D6050 R&DSystems IL-15 D1500 R&D Systems IL-22 D2200 R&D Systems LIF DLF00 R&DSystems FGF-21 DF2100 R&D Systems

Example 44: Transfection of Human Keratinocytes with RNA Encoding IL-15and/or IL-15RA

100,000 human epidermal keratinocytes (HEK, Gibco) were plated inEpiLife+Supplement S7. Cells were transfected according to Example 3with 2 μg of RNA encoding IL-15 or 1 μg each of RNA encoding IL-15 andRNA encoding IL-15RA. 24 hours after transfection, the medium wassampled and secreted IL-15 levels were measured using a human IL-15ELISA kit (D1500, R&D Systems) according the manufacturer'sinstructions. Secreted IL-15 levels were determined by measuring 450 nmabsorbance using a microplate reader (EMax Plus, Molecular Devices).Secreted IL-15 levels are shown in FIG. 35. As demonstrated in FIG. 35,co-transfection of IL-15 and IL-15RA significantly increased secretedIL-15 levels compared to transfection with IL-15 alone.

Example 45: Pharmacokinetic Study Via Intradermal Injection in Rats

Studies were conducted to evaluate the responses of Sprague Dawley ratsto intradermal administration of various RNAs. Specifically, 8-10 weeksold, female Sprague Dawley rats weighing about 200 g to about 350 g wereused for this study. A total of 33 rats were tested, and the animalswere assigned to study groups and treated as indicated in the Tablebelow:

Dose Dose Volume Number of Test Dose Dose Level Concentration (μL/perAnimals Group Group Color Article Route (μg) (μg/mL) injection)^(a)Females 1 White Control ID 4.0 20.0 200 3^(b) (NOVEPOEITIN) (4 × 50) 2Yellow TA1 ID 4.0 20.0 200 3^(b) (IL2) (4 × 50) 3 Green TA2 ID 4.0 20.0200 3^(b) (IL6) (4 × 50) 4 Blue TA3 ID 4.0 10.0 200 3^(b) (IL15) (4 ×50) 5 Red TA4 ID 4.0 20.0 200 3^(b) (IL15 + (10.0 each) (4 × 50) IL15RA)6 Dark Grey TA5 ID 4.0 20.0 200 3^(b) (IL22) (4 × 50) 7 Purple TA6 ID4.0 20.0 200 3^(b) (BMP2) (4 × 50) 8 Black TA7 ID 4.0 20.0 200 3^(b)(BDNF) (4 × 50) 9 White/Yellow TA8 ID 4.0 20.0 200 3^(b) (LIF) (4 × 50)10 Green/Blue TA9 ID 4.0 20.0 200 3^(b) (PTH) (4 × 50) 11 Red/Dark GreyTA10 ID 4.0 20.0 200 3^(b) (FGF21) (4 × 50) ^(a)Total dose volume(μL/per animal) was constant. Each animal received four intradermalinjections of 50 μL/per injection for a total of 200 μL per animal.^(b)Animals, euthanized on Day 3 without examination or necropsyIntradermal = ID

For the study, the animals were treated with 4 ug of RNA. All groupswere dosed via intradermal injection. Each dose was administered in fourintradermal injections of 50 μL/injection for a total of 200 uL peranimal. Injections occurred into previously marked sites near themidline of the dorsal lumbar area (upper left, upper right, lower leftand lower right quadrants). Dose time (after the last injection) wasrecorded. Additional markings were made as needed to allow foridentification of the dose site. Animals were administered with the RNAson day 1 and euthanized on day 3. Clinical observations were made on therats twice daily. Food consumption and body weight were also monitored.

During the study, approximately 1 ml of blood samples was collected fromthe jugular vein for pharmacokinetic analysis as follows:

Time Point PK^(a) Acclimation — Day 1 12 hours postdose Day 2 24 hourspostdose Day 3 48 hours postdose ^(a)Time points for blood collection (n= 3 animals/Group/Time point) PK = Pharmacokinetics

Results indicate that following administration of RNAs encoding FGF21,IL15, IL15 and IL15R, IL6, IL22, and NOVEPOEITIN, these proteins werereadily detected in the blood with protein levels peaking atapproximately 12 hours post injection (FIG. 39). Of note, the proteinstested in this study can be taken up by cells and tissues and/or canexert an effect near the site of expression without appreciableaccumulation in systemic circulation.

Example 46: Wound-Healing Therapy Comprising RNA Encoding IL22

Primary human epidermal keratinocytes (HEKn-APF) were plated in 6-wellplates coated with recombinant human Type-1 collagen at a density of200,000 cells/well in keratinocytes medium (EpiLife+Supplement S7).After 24 h, cells were transfected with 2 μg/well RNA encoding IL22synthesized according to Example 1 and 50 mM D-Glucose was added to thewells. The following day, a line was scratched in each well using aplastic 10 μL pipette tip. Wells were imaged at 0 h and 24 h afterscratching. The size of the scratch was measured using at both times,and healing was determined by calculating the ration of the size of thescratch at 24 h to the size of the scratch at 0 h (FIG. 40).

Example 47: A1AT Gene Editing and Repair in Human Cells

RNA encoding gene-editing proteins targeting the following sequences inthe A1AT gene was synthesized according to Example 1: L1:TAAGGCTGTGCTGACCATCG (SEQ ID NO: 611), R1: TAAAAACATGGCCCCAGCAG (SEQ IDNO: 612), and R2: TCTAAAAACATGGCCCCAGC (SEQ ID NO: 613). Cells were geneedited and gene-editing efficiency was measured according to Example 38(FIG. 41). Additionally, cells were co-transfected with gene editingproteins targeting sequences L1 and R1 and a repair template comprisingthe sequence:cccctccaggccgtgcataaggctgtgctgaccatcgacgtcaaagggactgaagctgctggggccatgtttttagaggcc(SEQ ID NO: 614), and gene-repair efficiency was measured according toExample 38, using the AatII enzyme (FIG. 42). Another sample of cellswas transfected with gene-editing proteins targeting the followingsequences in the A1AT gene: L2: TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615)and R3: TGTCTTCTGGGCAGCATCTC (SEQ ID NO: 616). Gene-editing efficiencywas measured according to Example 38 (FIG. 43).

Example 48: Alpha-1-Antitrypsin Deficiency Therapy Comprising RNAEncoding Gene-Editing Proteins and a Repair Template

RNA encoding gene-editing proteins targeting the following sequences: L:TAAGGCTGTGCTGACCATCG (SEQ ID NO: 611) and R: TAAAAACATGGCCCCAGCAG (SEQID NO: 612) and a repair template comprising the sequence: RT:cccctccaggccgtgcataaggctgtgctgaccatcgacgagaaagggactgaagctgctggggccatgtttttagaggcc(SEQ ID NO: 617) are formulated with a vehicle according to the methodsof the present invention and delivered to a subject afflicted with A1ATdeficiency by intraportal administration, resulting in correction of theZ mutation in the subject's liver cells, reduction of polymerized Zprotein accumulation in the subject's liver cells, increased secretionand serum levels of functional A1AT, and amelioration of one or more ofthe subject's symptoms.

Example 49: Friedrich's Ataxia Therapy Comprising RNA EncodingGene-Editing Proteins

RNA encoding one or more gene-editing proteins capable of creating oneor more double strand breaks in FXNA (SEQ ID NO: 582) and RNA encodingone or more gene-editing proteins capable of creating one or more doublestrand breaks in FXNB (SEQ ID NO: 583) are synthesized according toExample 1. RNA is formulated according to the methods of the presentinvention, and delivered to the heart of a patient with Friedrich'sataxia using a catheter. Target-sequence pairs are selected from: Pair1: TCCCACACGTGTTATTTGGC (SEQ ID NO: 618) and TGGCAACCAATCCCAAAGTT (SEQID NO: 619); Pair 2: TAATAAATAAAAATAAAAAA (SEQ ID NO: 620) andTTGCCTATTTTTCCAGAGAT (SEQ ID NO: 621).

Example 50: Alpha-1-Antitrypsin Deficiency Therapy Comprising RNAEncoding Gene-Editing Proteins

RNA encoding one or more gene-editing proteins capable of creating oneor more double strand breaks in A1AT_A (SEQ ID NO: 584) is synthesizedaccording to Example 1. RNA is formulated according to the methods ofthe present invention, and is delivered to the liver of a patient withA1AT deficiency by intraportal injection.

Example 51: Alpha-1-Antitrypsin Deficiency Therapy Comprising RNAEncoding Gene-Editing Proteins and a Short Repair Template

RNA encoding one or more gene-editing proteins capable of creating oneor more double strand breaks in A1AT_B (SEQ ID NO: 585) is synthesizedaccording to Example 1. RNA and a single stranded DNA sequencecontaining the sequence: A1AT_RT (SEQ ID NO: 586) are formulatedaccording to the methods of the present invention, and are delivered tothe liver of a patient with A1AT deficiency by intraportal injection.

Example 52: Gene-Editing Proteins Comprising DNA-Binding and DeaminaseActivity

RNA encoding a gene-editing protein comprising two or more repeatsequences, followed by: BASE_EDIT_FRONT (SEQ ID NO: 587), followed byany of: BASE_EDIT_ADA1 (SEQ ID NO: 588), BASE_EDIT_ADA2 (SEQ ID NO:589), BASE_EDIT_ADA3 (SEQ ID NO: 590), BASE_EDIT_ADA4 (SEQ ID NO: 591),BASE_EDIT_CDA1 (SEQ ID NO: 592) and BASE_EDIT_CDA2 (SEQ ID NO: 593) issynthesized according to Example 1. The gene-editing protein is capableof correcting one or more mutations within 1 to 50 bases downstream ofthe target sequence.

Example 53: Gene Editing of A1AT, COL7A1, HBB, and PD-1

With reference to Example 1, Table 4, the following templates andtargets were used:

Template Target Sequence SEQ ID NO: A1AT TALEN L TGCCTGGTCCCTGTCTCCCT615 A1AT TALEN R TGTCTTCTGGGCAGCATCTC 616 A1AT RIBOSLICE L_ATGCCTGGTCCCTGTCTCCCT 615 A1AT RIBOSLICE L_B TGCCTGGTCCCTGTCTCCCT 615A1AT RIBOSLICE R_A TGTCTTCTGGGCAGCATCTC 616 A1AT RIBOSLICE R_BTGTCTTCTGGGCAGCATCTC 616 COL7A1 exon 73 TALEN L TATTCCCGGGCTCCCAGGCA 622COL7A1 exon 73 TALEN R TCTCCTGGCCTTCCTGCCTC 612 COL7A1 exon 73 rs3L 50ATATTCCCGGGCTCCCAGGCA 622 COL7A1 exon 73 rs3L 50B TATTCCCGGGCTCCCAGGCA622 COL7A1 exon 73 rs3R 50A TCTCCTGGCCTTCCTGCCTC 612COL7A1 exon 73 rs3R 50B TCTCCTGGCCTTCCTGCCTC 612COL7A1 exon 73 TALEN L EA TATTCCCGGGCTCCCAGGCA 622COL7A1 exon 73 TALEN L Het TATTCCCGGGCTCCCAGGCA 622COL7A1 exon 73 TALEN R EA TCTCCTGGCCTTCCTGCCTC 612COL7A1 exon 73 TALEN R Het TCTCCTGGCCTTCCTGCCTC 612COL7A1 exon 73 TALEN L EA/Het TATTCCCGGGCTCCCAGGCA 622COL7A1 exon 73 TALEN R EA/Het TCTCCTGGCCTTCCTGCCTC 612HBB exon 1 TALEN L TGGTGCATCTGACTCCTGAG 623 HBB exon 1 TALEN RTCACCTTGCCCCACAGGGCA 624 PD-1 exon 1 TALEN L TCCAGGCATGCAGATCCCAC 625PD-1 exon 1 TALEN R TTGTAGCACCGCCCAGACGA 626 TTR (forward (top)TGCTGTCCGAGGCAGTCCTG 639; 640 and reverse (bottom) TCAGCAGCCTTTCTGAACACTTR (forward (top) TGTCCGAGGCAGTCCTGCCA 641; 642 and reverse (bottom)TCAGCAGCCTTTCTGAACAC TTR (forward (top) TCCGAGGCAGTCCTGCCATC 643; 644and reverse (bottom) TCAGCAGCCTTTCTGAACAC TTR (forward (top)TGTCCGAGGCAGTCCTGCCA 645; 646 and reverse (bottom) TCATCAGCAGCCTTTCTGAATTR (forward (top) TCCGAGGCAGTCCTGCCATC 647; 648 and reverse (bottom)TCATCAGCAGCCTTTCTGAA TTR (forward (top) TCCGAGGCAGTCCTGCCATC 649; 650and reverse (bottom) TGTCATCAGCAGCCTTTCTG

About 100,000 primary human neonatal epidermal keratinocytes were platedin Epi Life+Supplement S7 and transfected according to Example 3 withRNA encoding gene-editing proteins that target the sequences L:TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615) and R: TGTCTTCTGGGCAGCATCTC (SEQID NO: 616), which were located approximately 75 bp from theAlpha-1-Antitrypsin (A1AT) start codon. Cells were gene edited, andgene-editing efficiency was measured as previously described. Results asshown in FIG. 46 demonstrate efficient gene-editing by the gene editingproteins.

About 100,000 primary human neonatal epidermal keratinocytes were platedin EpiLife+Supplement S7 and transfected according to Example 3 with RNAencoding gene-editing proteins that target the sequences L:TATTCCCGGGCTCCCAGGCA (SEQ ID NO: 622) and R: TCTCCTGGCCTTCCTGCCTC (SEQID NO: 612), which were located near the end of exon 73 of COL7A1. Cellswere gene edited, and gene-editing efficiency was measured as previouslydescribed. Results as shown in FIG. 47 demonstrate efficientgene-editing by the gene editing proteins.

About 50,000 primary human neonatal epidermal keratinocytes were platedin EpiLife+Supplement S7 and transfected according to Example 3 with RNAencoding various gene-editing proteins that target the sequences L:TATTCCCGGGCTCCCAGGCA (SEQ ID NO: 622) and R: TCTCCTGGCCTTCCTGCCTC (SEQID NO: 612), which were located near the end of exon 73 of COL7A1. Inparticular, the gene-editing proteins comprised the endonucleasecleavage domain of FokI and variants thereof. The variants included aFokI variant with enhanced activity (S35P, K58E), a FokI heterodimer(i.e., L: Q103E, N113D, I116L; and R: E107K, H154R, 1155K), and acombination thereof (i.e., L: S35P, K58E, Q103E, N113D, I116L; and R:S35P, K58E, E107K, H154R, 1155K). Cells were gene edited, andgene-editing efficiency was measured as previously described. Results asshown in FIG. 48 demonstrate efficient gene-editing by the gene editingproteins.

About 100,000 primary human neonatal epidermal keratinocytes(animal-protein free) were plated in EpiLife+Supplement S7. Cells weretransfected according to Example 3 with 2 μg RNA encoding the HBB exon 1TALEN L and HBB exon 1 TALEN R gene-editing proteins (1 μg each). 48hours after transfection, DNA was harvested and analyzed for geneediting (T7E1 assay; forward primer: gccaaggacaggtacggctgtcatc (SEQ IDNO: 627); reverse primer: cttgccatgagccttcaccttagggttg (SEQ ID NO: 628);product size: 518nt; predicted band sizes: 300nt, 218nt). Results asshown in FIG. 61 demonstrate efficient gene-editing by the gene editingproteins.

About 100,000 primary human neonatal epidermal keratinocytes(animal-protein free) were plated in EpiLife+Supplement S7. Cells weretransfected according to Example 3 with 2 μg RNA encoding the PD1 exon 1TALEN L and PD1 exon 1 TALEN R gene-editing proteins (1 μg each). After48 hours, DNA was harvested and analyzed for gene editing (T7E1 assay;forward primer: tcctctgtctccctgtctctgtctctctctc (SEQ ID NO: 594);reverse primer: ggacttgggccaggggaggag (SEQ ID NO: 595); product size:612nt; predicted band sizes: 349nt, 263nt). Results as shown in FIG. 62demonstrate efficient gene-editing by the gene editing proteins.

Example 54: Transfection of Primary Human Keratinocytes with RNAEncoding GDF15, IFKB, KRT5, KRT14, SOD3, or hESM1

As shown in FIG. 49, 100,000 primary human neonatal epidermalkeratinocytes were plated in EpiLife+Supplement S7. Cells weretransfected according to Example 3 with 2 μg of RNA encoding hGDF15.Following transfection, the culture media was sampled at various timepoints and analyzed for hGDF15 levels using ELISA (R&D DGD150) accordingto manufacturer's instructions. FIG. 49 demonstrates that hGDF15 levelswere upregulated in a time-dependent manner following transfection.

As shown in FIG. 50, 100,000 primary human neonatal epidermalkeratinocytes were plated in EpiLife+Supplement S7. Cells weretransfected according to Example 3 with 2 μg of RNA encoding IFKB or anIFKB variant (S32A and S36A). Following transfection, the culture mediawas sampled at various time points and analyzed for IFKB levels usingELISA (Abcam, ab176644) according to manufacturer's instructions. FIG.50 demonstrates increased levels IFKB or the IFKB variant followingtransfection.

As shown in FIG. 51, 100,000 primary human neonatal epidermalkeratinocytes were plated in 6-well plates in EpiLife+Supplement S7.Cells were transfected according to Example 3 with 2 μg of RNA encodingKRT5 or KRT14 (or GFP tagged versions of KRT5 or KRT14). Followingtransfection, GFP imaging was carried out to determine KRT5 or KRT14expression. FIG. 51 demonstrates efficient transfection and increasedlevels KRT5-GFP or KRT14-GFP following transfection.

As shown in FIG. 52, 20,000 primary human neonatal epidermalkeratinocytes were plated in 24-well plates in EpiLife+Supplement S7.Cells were transfected according to Example 3 with 0.2 μg of RNAencoding SOD3. Following transfection, cells were fixed and stained for24 hours with a 1:100 dilution of NBP2-38493 (Novus) rabbit anti humanSOD3 primary antibody and a 1:1000 dilution of 488 donkey anti-rabbitsecondary antibody. Results demonstrate efficient transfection androbust levels of SOD3 in transfected cells.

As shown in FIG. 60, 200,000 primary human neonatal epidermalkeratinocytes (animal-protein free) were plated in 6-well plates inEpiLife+Supplement S7. Cells were transfected according to Example 3with 2 μg RNA encoding hESM1. 52 hours after transfection, the culturemedium was analyzed for ESM1 by ELISA (Abcam ab213776). Resultsdemonstrate efficient transfection and robust levels of hESM1 intransfected cells.

Example 55: In Vivo Targeting of GDF15 (MIC1)

Studies were conducted to evaluate the responses of Zucker-obese (ZDF)rats to administration of RNA encoding hGDF15. The Zucker-obese rats(ZDF) are a well-studied obesity model that mimics many aspects of thehuman disease. Specifically, 8-10 weeks old male rats were used for thisstudy. A total of 15 ZDF and 15 wildtype control Sprague-Dawley ratswere tested, and the animals were assigned to study groups and treatedas indicated in the Table below:

Dose Group Rat Test Level Concentration Volume^(a) Number Group ColorSpecies Article Route (μg) (μg/mL) (μL) of Males 1 White Sprague-Control Intradermal 0 0 50 per site; 5 Dawley 500 total 2 YellowSprague- FG-02 10X 10 50 per site; 10 Dawley (i.e., RNA (0.5 μg/ 500total encoding injection; 5 μg GDF15) total) 3 Green ZDF Control 0 0 50per site; 5 500 total 4 Blue ZDF FG-02 10X 10 50 per site; 10 (i.e., RNA(0.5 μg/ 500 total encoding injection; 5 μg GDF15) total) ^(a)Note:Total dose volume (μL/per animal) was constant

Groups 1 to 4 were dosed by intradermal (ID) injection. Each dose wasadministered on the back in ten ID injections of 50 μL/injection for atotal of 500 uL per animal.

Each dose site was as follows:

Day  1  dose   Day  8  dose  Head————————-Spine————————————Tail Day  15  dose  Day  22  dose  

RNA or control was administered once weekly (Days 1, 8, and 15), withthe first dose being on Day 1, and the last dose on Day 15. Animals wereeuthanized, without examination on Day 44.

Blood was collected from the rats according to the schedule below andused for serum chemistry and pharmacokinetics analysis:

Time Point (Study Week) Serum Chemistry PK Acclimation — — (Week −2)Dosing 2x Day 1 (1x), 2 (1x), 3 (1x), 4 (1x), (Week 1) and 5 (1x) Dosing2x Day 9^(c) (Week 2) Dosing 2x Day 16^(c) (Week 3) Dosing 2x Day 23(Week 4) Dosing 2x — (Week 5) Dosing 2x — (Week 6) Dosing 1x — (Week 7)— = Not applicable PK = Pharmacokinetics x = Number of times procedureperformed ^(c)Sample collected 24 hours postdose

Serum chemistry analysis was carried out using an AU680 analyzer for thefollowing: Albumin, Alkaline Phosphatase, Alanine Aminotransferase,Aspartate Aminotransferase, Total Bilirubin, Calcium, Total Cholesterol,Creatine Kinase, Creatinine, Glucose, Inorganic Phosphorus, TotalProtein, Triglyceride, Sodium, Potassium, Chloride, Globulin,Albumin/Globulin Ratio, and Blood Urea Nitrogen. Results for changes inthe serum levels of ALT, AST, total cholesterol, triglycerides, andglucose are provided in FIGS. 53-58. As shown in the FIG. 58,administration of RNA encoding GDF15 into ZDF rats resulted in reducedALT, AST, and Glucose levels but elevated total cholesterol andtriglyceride levels in the serum. These results suggest that diabeticobese animals treated with RNA encoding GDF15 exhibited improved liverhealth, as indicated by decreased serum levels of ALT and AST, improved(lower) blood glucose levels, and lipid mobilization, as indicated byincreased serum cholesterol and triglyceride levels. These resultstherefore suggest that RNA encoding GDF15 may be used to treat, interalia, diabetes, obesity, fatty liver, and other diseases that includeliver inflammation, elevated blood glucose, and/or abnormal lipidmetabolism.

The serum levels of GDF15 in the treated rats were confirmed as shown inFIG. 59. As shown, robust GDF15 levels were observed even at day 16(i.e., day 15+24 h) in both ZDF and control Sprague-dawley rats injectedwith RNA encoding GDF15.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

What is claimed is:
 1. A method for treating a metabolic disorder,comprising administering an effective amount of a synthetic RNA encodinggrowth differentiation factor 15 (GDF15) or endothelial cell specificmolecule 1 (ESM1) to a subject in need thereof, wherein the syntheticRNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.
 2. The method of claim 1, wherein themetabolic disorder is selected from Type I diabetes, Type II diabetes,insulin resistance, obesity, dyslipidemia, hypercholesterolemia,hyperglycemia, hyperinsulinemia, hypertension, hepatosteaotosis such asnon-alcoholic steatohepatitis (NASH) and non-alcoholic fatty acid liverdisease (NAFLD), cancer, a disease or disorder associated with impairedlipid metabolism, a disease or disorder associated with impaired renalfunction, a disease or disorder associated with impaired hepaticfunction, a disease or disorder associated with impaired lung function,a vascular or cardiovascular disease or disorder, muscle wasting,inflammation, and a respiratory disease.
 3. The method of claim 2,wherein the disease or disorder associated with impaired renal functionis selected from chronic kidney diseases, acute kidney injury,nephropathy, diabetic nephropathy, kidney failure, and kidney fibrosis.4. The method of claim 2, wherein the vascular or cardiovascular diseaseor disorder is selected from coronary artery disease, cardiomyopathy,hypertension, atrial fibrillation, preeclampsia, peripheral arterydisease, atherosclerosis, heart failure, acute myocardial infarction,acute coronary syndrome, muscle wasting, hypertensive ventricularhypertrophy, hypertensive cardiomyopathy, ischemic heart disease,myocardial infarction, abdominal aortic aneurysm, a blood clot, deepvein thrombosis, venous stasis disease, phlebitis, and varicose veins.5. The method of any one of claims 1 to 4, wherein the treatment resultsin reduction in one or more of aspartate transaminase (AST), alaninetransaminase (ALT), or glucose levels in the subject, as compared tountreated subjects.
 6. The method of any one of claims 1 to 5, whereinthe treatment results in increased lipid mobilization in the subject, ascompared to untreated subjects.
 7. The method of any one of claims 1 to6, wherein the administration is intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intracerebral, intravaginal, transdermal, intraportal,rectally, by inhalation, or topically.
 8. A method for modulating GDF15,comprising administering an effective amount of a synthetic RNA encodingGDF15 to a subject, wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity. 9.The method of claim 8, wherein the modulating results in reduction inone or more of ALT, AST, or glucose levels in the subject, as comparedto untreated subjects.
 10. The method of claim 8 or claim 9, wherein themodulating results in increased lipid mobilization in the subject, ascompared to untreated subjects.
 11. The method of any one of claims 8 to10, wherein the administration is intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intracerebral, intravaginal, transdermal, intraportal,rectally, by inhalation, or topically.
 12. The method of any one ofclaims 8 to 11, wherein the modulating results in an increase in thequantity of GDF15 in the subject.
 13. The method of any one of claims 8to 11, wherein the modulating results in a decrease in the quantity ofGDF15 in the subject.
 14. A method for modulating ESM1, comprisingadministering an effective amount of a synthetic RNA encoding ESM1 to asubject, wherein the synthetic RNA comprises one or more non-canonicalnucleotides that avoid substantial cellular toxicity.
 15. The method ofclaim 14, wherein the modulating results in reduction in one or more ofALT, AST, or glucose levels in the subject, as compared to untreatedsubjects.
 16. The method of claim 14 or claim 15, wherein the modulatingresults in increased lipid mobilization in the subject, as compared tountreated subjects.
 17. The method of any one of claims 14 to 16,wherein the administration is intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intracerebral, intravaginal, transdermal, intraportal,rectally, by inhalation, or topically.
 18. The method of any one ofclaims 14 to 17, wherein the modulating results in an increase in thequantity of ESM1 in the subject.
 19. The method of any one of claims 14to 17, wherein the modulating results in a decrease in the quantity ofESM1 in the subject.
 20. A method for treating a liver disordercomprising administering an effective amount of a synthetic RNA encodinggrowth differentiation factor 15 (GDF15) or endothelial cell specificmolecule 1 (ESM1) to a subject in need thereof, wherein: the syntheticRNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity; and the treatment reduces one or more offatty liver, NAFLD, NASH, inflammation, hepatitis, fibrosis, cirrhosis,and hepatocellular carcinoma in the subject.
 21. The method of claim 20,wherein the treatment results in reduction in one or more of AST, ALT,or glucose levels in the subject, as compared to untreated subjects. 22.The method of claim 20 or claim 21, wherein the treatment results inincreased lipid mobilization in the subject, as compared to untreatedsubjects.
 23. The method of any one of claims 20 to 23, wherein theadministration is directed to the liver.
 24. The method of any one ofclaims 20 to 24, wherein the administration is intraportal injection.25. A method for treating Friedrich's Ataxia comprising administering toa subject in need thereof an effective amount of (a) a synthetic RNAencoding a gene-editing protein capable of creating a double strandbreak in FXNA (SEQ ID NO: 582), and/or (b) a synthetic RNA encoding agene-editing protein capable of creating a double strand break in FXNB(SEQ ID NO: 583), wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity. 26.The method of claim 25, wherein the treatment comprises administering toa subject in need thereof an effective amount of (a) the synthetic RNAencoding the gene-editing protein capable of creating a double strandbreak in FXNA (SEQ ID NO: 582), and (b) the synthetic RNA encoding thegene-editing protein capable of creating a double strand break in FXNB(SEQ ID NO: 583).
 27. The method of claim 25 or claim 26, wherein theadministration is directed to the heart.
 28. The method of any one ofclaims 25 to 27, wherein the administration is via a catheter.
 29. Themethod of any one of claims 25 to 28, wherein the treatment amelioratesone or more of muscle weakness, loss of coordination, vision impairment,hearing impairment, slurred speech, scoliosis, pes cavus deformity ofthe foot, diabetes, and heart disorders, selected from one or moreatrial fibrillation, tachycardia and hypertrophic cardiomyopathy. 30.The method of any one of claims 25 to 29, wherein the gene-editingprotein is selected from a CRISPR/Cas9, TALEN and a zinc fingernuclease.
 31. The method of any one of claims 25 to 29, wherein thegene-editing protein comprises: (a) a DNA-binding domain comprising aplurality of repeat sequences and at least one of the repeat sequencescomprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629)and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E,“w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, ornull, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (b) a nuclease domaincomprising a catalytic domain of a nuclease.
 32. The method of claim 31,wherein the nuclease domain is capable of forming a dimer with anothernuclease domain.
 33. The method of claim 31 or claim 32, wherein thenuclease domain comprises the catalytic domain of a protein comprisingthe amino acid sequence of SEQ ID NO:
 632. 34. A method for reducinginflammation, comprising administering an effective amount of asynthetic RNA encoding superoxide dismutase 3 (SOD3) or IF_(κ)B, whereinthe synthetic RNA comprises one or more non-canonical nucleotides thatavoid substantial cellular toxicity to a subject in need thereof. 35.The method of claim 34, wherein the inflammation is associated with alung disease or disorder.
 36. The method of claim 34 or claim 35,wherein the lung disease or disorder is selected from Asbestosis,Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic ObstructivePulmonary Disease (COPD), Common Cold, Croup, Cystic Fibrosis,Hantavirus, Idiopathic Pulmonary Fibrosis, Influenza, Lung Cancer,Pandemic Flu, Pertussis, Pleurisy, Pneumonia, Pulmonary Embolism,Pulmonary Hypertension, Respiratory Syncytial Virus (RSV), Sarcoidosis,Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS), andTuberculosis.
 37. The method of claim 36, wherein the inflammation isassociated with a lung infection.
 38. The method of claim 37, whereinthe lung infection is cause by a bacterium, a fungus, a protozoa, amulti-cellular organism, a particulate, or a virus.
 39. The method ofany one of claims 34 to 38, wherein the synthetic RNA is administered byinhalation.
 40. The method of any one of claims 34 to 39, wherein theinflammation is associated with sepsis.
 41. A method for treatingalpha-1 antitrypsin (A1AT) deficiency comprising administering to asubject in need thereof an effective amount of (a) a synthetic RNAencoding a gene-editing protein capable of creating a double strandbreak in A1AT_A (SEQ ID NO: 584) and/or (b) a synthetic RNA encoding agene-editing protein capable of creating a double strand break in A1AT_B(SEQ ID NO: 585), wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity. 42.The method of claim 41, wherein the treatment comprises administering toa subject in need thereof an effective amount of (a) the synthetic RNAencoding the gene-editing protein capable of creating a double strandbreak in A1AT_A (SEQ ID NO: 584) and (b) the synthetic RNA encoding thea gene-editing protein capable of creating a double strand break inA1AT_B (SEQ ID NO: 585).
 43. The method of claim 41 or claim 42, whereinthe administration is directed to the liver.
 44. The method of any oneof claims 41 to 43, wherein the administration is intraportal injection.45. The method of any one of claims 41 to 44, wherein the treatmentcorrects the Z mutation in the subject's liver cells.
 46. The method ofany one of claims 41 to 45, wherein the treatment reduces polymerized Zprotein accumulation in the subject's liver cells.
 47. The method of anyone of claims 41 to 46, wherein the treatment increases secretion and/orserum levels of functional A1AT.
 48. The method of any one of claims 41to 47, wherein the treatment ameliorates one or more of chronic cough,emphysema, COPD, liver failure, hepatitis, hepatomegaly, jaundice, andcirrhosis.
 49. The method of any one of claims 41 to 48, wherein thegene-editing protein is selected from a CRISPR/Cas9, TALEN, and a zincfinger nuclease.
 50. The method of any one of claims 41 to 48, whereinthe gene-editing protein comprises: (a) a DNA-binding domain comprisinga plurality of repeat sequences and at least one of the repeat sequencescomprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629)and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E,“w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, ornull, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (b) a nuclease domaincomprising a catalytic domain of a nuclease.
 51. The method of claim 50,wherein the nuclease domain is capable of forming a dimer with anothernuclease domain.
 52. The method of claim 50 or claim 51, wherein thenuclease domain comprises the catalytic domain of a protein comprisingthe amino acid sequence of SEQ ID NO:
 632. 53. A method for treatingalpha-1 antitrypsin (A1AT) deficiency comprising administering aneffective amount of a synthetic RNA encoding A1AT to a subject, whereinthe synthetic RNA comprises one or more non-canonical nucleotides thatavoid substantial cellular toxicity.
 54. The method of claim 53, whereinthe administration is directed to the liver.
 55. The method of claim 53or claim 54, wherein the administration is intraportal injection. 56.The method of any one of claims 53 to 55, wherein the treatment correctsthe Z mutation in the subject's liver cells.
 57. The method of any oneof claims 53 to 56, wherein the treatment reduces polymerized Z proteinaccumulation in the subject's liver cells.
 58. The method of any one ofclaims 53 to 57, wherein the treatment increases secretion and/or serumlevels of functional A1AT.
 59. The method of any one of claims 53 to 58,wherein the treatment ameliorates one or more of the subject's symptoms.60. A method for reversing an alpha-1 antitrypsin (A1AT) deficiency in acell comprising (a) obtaining a cell comprising a defective A1AT gene;and (b) contacting the cell with a synthetic RNA encoding A1AT, whereinthe synthetic RNA comprises one or more non-canonical nucleotides thatavoid substantial cellular toxicity.
 61. The method of claim 60, furthercomprising steps of (c) obtaining the cell contacted in step (b); and(d) administering the cell to a subject in need thereof.
 62. The methodof claim 60 or claim 61, wherein the administration is directed to theliver.
 63. The method of any one of claims 60 to 62, wherein theadministration is intraportal injection.
 64. A method for reversing analpha-1 antitrypsin (A1AT) deficiency in a cell comprising (a) obtaininga cell comprising a defective A1AT gene; and (b) contacting the cellwith one or both of a synthetic RNA encoding a gene-editing proteincapable of creating a double strand break in A1AT_A (SEQ ID NO: 584) anda synthetic RNA encoding a gene-editing protein capable of creating adouble strand break in A1AT_B (SEQ ID NO: 585), wherein the syntheticRNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.
 65. The method of claim 64, furthercomprising steps of (c) obtaining the cell contacted in step (b); and(d) administering the cell to a subject in need thereof.
 66. The methodof claim 64 or claim 65, wherein the administration is directed to theliver.
 67. The method of any one of claims 64 to 66, wherein theadministration is intraportal injection.
 68. The method of any one ofclaims 64 to 67, wherein the gene-editing protein is selected from aCRISPR/Cas9, TALEN and a zinc finger nuclease.
 69. The method of any oneof claims 64 to 67, wherein the gene-editing protein comprises: (a) aDNA-binding domain comprising a plurality of repeat sequences and atleast one of the repeat sequences comprises the amino acid sequence:LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acidslong, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y”is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (b) a nucleasedomain comprising a catalytic domain of a nuclease.
 70. The method ofclaim 69, wherein the nuclease domain is capable of forming a dimer withanother nuclease domain.
 71. The method of claim 69 or claim 70, whereinthe nuclease domain comprises the catalytic domain of a proteincomprising the amino acid sequence of SEQ ID NO:
 632. 72. A method formodulating BMP7, comprising administering an effective amount of asynthetic RNA encoding BMP7 to a subject, wherein the synthetic RNAcomprises one or more non-canonical nucleotides that avoid substantialcellular toxicity.
 73. The method of claim 72, wherein the modulatingresults in an increase in the quantity of BMP7 in the subject.
 74. Themethod of claim 72 or claim 73, wherein the modulating results in adecrease in the quantity of BMP7 in the subject.
 75. The method of claim72 or claim 73, wherein the modulating results in an increase inintracellular alkaline phosphatase activity.
 76. The method of any oneof claims 72 to 75 wherein the synthetic RNA encoding BMP7 comprises asequence encoding the BMP7 signal peptide.
 77. The method of claim 76,wherein the BMP7 signal peptide comprises the sequence of SEQ ID NO:597.
 78. The method of any one of claims 72 to 77, wherein theadministration is directed to the liver.
 79. The method of any one ofclaims 72 to 78, wherein the administration is intraportal injection.80. The method of any one of claims 72 to 79, wherein the administrationtreats diabetic nephropathy.
 81. The method of any one of claims 72 to80, wherein the administration treats liver fibrosis.
 82. The method ofany one of claims 72 to 77, wherein the administration is directed tothe kidney.
 83. The method of claim 82, wherein the administration isintravenous or intradermal.
 84. The method of claim 82 or claim 83,wherein the administration treats kidney disease.
 85. The method of anyone of claims 82 to 84, wherein the kidney disease is diabeticnephropathy.
 86. The method of any one of claims 72 to 85, wherein themodulating results in reduced levels of urine albumin in the subject.87. A method for modulating an immune checkpoint molecule, comprisingadministering an effective amount of a synthetic RNA encoding the immunecheckpoint molecule to a subject, wherein the synthetic RNA comprisesone or more non-canonical nucleotides that avoid substantial cellulartoxicity.
 88. A method for treating a subject, comprising administeringa synthetic RNA encoding a gene-editing protein targeting an immunecheckpoint molecule gene.
 89. A method for treating a subject comprising(a) obtaining a cell comprising an immune checkpoint molecule gene and(b) contacting the cell with a synthetic RNA encoding a gene-editingprotein capable of creating a double strand break in the immunecheckpoint molecule gene, wherein the synthetic RNA comprises one ormore non-canonical nucleotides that avoid substantial cellular toxicity.90. The method of claim 89, further comprising steps of (c) obtainingthe cell contacted in step (b); and (d) administering the cell to asubject in need thereof.
 91. The method of any one of claims 88 to 90,wherein the gene-editing protein is selected from a CRISPR/Cas9, TALENand a zinc finger nuclease.
 92. The method of any one of claims 88 to90, wherein the gene-editing protein comprises: (a) a DNA-binding domaincomprising a plurality of repeat sequences and at least one of therepeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG(SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v”is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H,K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) orGGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (b) a nuclease domaincomprising a catalytic domain of a nuclease.
 93. The method of claim 92,wherein the nuclease domain is capable of forming a dimer with anothernuclease domain.
 94. The method of claim 92 or claim 93, wherein thenuclease domain comprises the catalytic domain of a protein comprisingthe amino acid sequence of SEQ ID NO:
 632. 95. The method of any one ofclaims 87 to 94, wherein the immune checkpoint molecule is selected fromPD-1, PD-L1, PD-L2, CTLA-4, ICOS, LAG3, OX40, OX40L, and TIM3.
 96. Themethod of claim 95, wherein the immune checkpoint molecule is PD-1. 97.The method of any one of claims 87 to 96, wherein the administeringstimulates or enhances an immune response in the subject.
 98. The methodof any one of claims 87 to 96, wherein the administering inhibits orreduces an immune response in the subject.
 99. The method of any one ofclaims 87 to 98, wherein the subject is afflicted with a cancer. 100.The method of any one of claims 87 to 98, wherein the subject isafflicted with an autoimmune disease.
 101. The method of any one ofclaims 87 to 100, wherein the administration is intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intracerebral, intravaginal, transdermal,intraportal, rectally, by inhalation, or topically.
 102. The method ofany one of claims 87 to 101, wherein the modulating results in anincrease in the quantity of PD-1 in the subject.
 103. The method of anyone of claims 87 to 101, wherein the modulating results in a decrease inthe quantity of PD-1 in the subject.
 104. A method for treating a cancercomprising: (a) isolating an cell from a subject, the cell being animmune cell or hematopoietic cell; (b) contacting the isolated cell withan effective amount of a synthetic RNA encoding a chimeric antigenreceptor (CAR), wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity; and(c) administering the cell to the subject.
 105. The method of claim 104,wherein the immune cell is a T cell.
 106. A method for making a chimericantigen receptor (CAR) T cell, comprising: (a) obtaining a cell from asubject; and (b) contacting the cell with a synthetic RNA encoding agene-editing protein capable of creating a double strand break to yielda safe harbor locus for CAR insertion; wherein the synthetic RNAcomprises one or more non-canonical nucleotides that avoid substantialcellular toxicity.
 107. The method of claim 106, wherein thegene-editing protein is selected from a CRISPR/Cas9, TALEN and a zincfinger nuclease.
 108. The method of claim 106, wherein the gene-editingprotein comprises: (a) a DNA-binding domain comprising a plurality ofrepeat sequences and at least one of the repeat sequences comprises theamino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between36 and 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N,“x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” isGGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO:631); and (b) a nuclease domain comprising a catalytic domain of anuclease.
 109. The method of claim 108, wherein the nuclease domain iscapable of forming a dimer with another nuclease domain.
 110. The methodof claim 108 or claim 109, wherein the nuclease domain comprises thecatalytic domain of a protein comprising the amino acid sequence of SEQID NO:
 632. 111. A method for increasing the persistence of a chimericantigen receptor (CAR) T cell, comprising contacting the chimericantigen receptor (CAR) T cell with a synthetic RNA encoding a encoding atelomerase, wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity. 112.A method for treating a skin wound, comprising administering aneffective amount of a synthetic RNA encoding interleukin 22 (IL22) to asubject, wherein the synthetic RNA comprises one or more non-canonicalnucleotides that avoid substantial cellular toxicity.
 113. The method ofclaim 112, wherein the administration is directed to a population ofcells of the integumentary system.
 114. The method of claim 113, whereinthe population of cells comprises one or more of cells of the epidermis,cells of the basement membrane, cells of the dermis, and/or cells of thesubcutis.
 115. The method of claim 113 or claim 114, wherein thepopulation of cells are cells of the epidermis, and comprise one more ofcells of the stratum corneum, stratum lucidum, stratum granulosum,stratum spinosum, and/or stratum germinativum.
 116. The method of anyone of claims 113 to 115, wherein the population of cells is cells ofthe dermis, and comprise one or more of cells from the papillary regionand the reticular region.
 117. The method of any one of claims 112 to116, wherein the administration is by subcutaneous injection,intradermal injection, subdermal injection, intramuscular injection, ortopical administration.
 118. The method of claim 117, wherein theadministration is intradermal injection to one or more of the dermis orepidermis.
 119. The method of any one of claims 112 to 118, wherein theeffective amount is from about 10 ng to about 5000 ng per treatment areaof about 10 cm² or less, or about 5 cm² or less, or about 1 cm² or less,or about 0.5 cm² or less, or about 0.2 cm² or less.
 120. The method ofany one of claims 112 to 119, wherein about 10 ng, or about 20 ng, orabout 50 ng, or about 100 ng, or about 200 ng, or about 300 ng, or about400 ng, or about 500 ng, or about 600 ng, or about 700 ng, or about 800ng, or about 900 ng, or about 1000 ng, or about 1100 ng, or about 1200ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000ng, or about 3000 ng, or about 4000 ng, or about 5000 ng of thesynthetic RNA is administered per treatment area of about 10 cm² orless, or about 5 cm² or less, or about 1 cm² or less, or about 0.5 cm²or less, or about 0.2 cm² or less.
 121. The method of any one of claims112 to 120, wherein the modulating results in an increase in thequantity of IL22 in the subject.
 122. The method of any one of claims112 to 120, wherein the modulating results in a decrease in the quantityof IL22 in the subject.
 123. The method of any one of claims 112 to 122,wherein the effective amount of synthetic RNA is administered using anarray of needles covering an affected area of the subject.
 124. Themethod of any one of claims 112 to 123, wherein a treatment area ismechanically massaged after administration.
 125. The method of any oneof claims 112 to 124, wherein a treatment area is exposed to electricpulses after administration.
 126. The method of claim 125, wherein theelectric pulses are between about 10V and about 200V for from about 50microseconds to about 1 second.
 127. The method of claim 125 or claim126, wherein the electric pulses are generated around the treatment areaby a multielectrode array.
 128. A method for treating a metabolicdisorder, comprising administering a synthetic RNA encoding agene-editing protein targeting a defective gene, wherein theadministration is by intraportal injection.
 129. A method for treating ametabolic disorder comprising (a) obtaining a cell comprising adefective gene and (b) contacting the cell with a synthetic RNA encodinga gene-editing protein capable of creating a double strand break in thedefective gene, wherein the synthetic RNA comprises one or morenon-canonical nucleotides that avoid substantial cellular toxicity. 130.The method of claim 129, further comprising steps of (c) obtaining thecell contacted in step (b); and (d) administering the cell to a subjectin need thereof by intraportal injection.
 131. The method of any one ofclaims 128 to 130, wherein the gene-editing protein is selected from aCRISPR/Cas9, TALEN and a zinc finger nuclease.
 132. The method of anyone of claims 128 to 130, wherein the gene-editing protein comprises:(a) a DNA-binding domain comprising a plurality of repeat sequences andat least one of the repeat sequences comprises the amino acid sequence:LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acidslong, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y”is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (b) a nucleasedomain comprising a catalytic domain of a nuclease.
 133. The method ofclaim 132, wherein the nuclease domain is capable of forming a dimerwith another nuclease domain.
 134. The method of claim 132 or claim 133,wherein the nuclease domain comprises the catalytic domain of a proteincomprising the amino acid sequence of SEQ ID NO:
 632. 135. The method ofany one of claims 128 to 134, wherein the metabolic disorder is selectedfrom a disorder of carbohydrate metabolism, a disorder of amino acidmetabolism, a disorder of the urea cycle, a disorder of fatty acidmetabolism, a disorder of porphyrin metabolism, a disorder of lysosomalstorage, a disorder of peroxisome biogenesis, and a disorder of purineor pyrimidine metabolism.
 136. The method of claim 135, wherein themetabolic disorder is a disorder of carbohydrate metabolism and whereinthe disease is galactosemia and the defective gene is optionally GALT,GALK1, or GALE; wherein the disease is essential fructosuria and thedefective gene is optionally KHK; wherein the disease is Hereditaryfructose intolerance and the defective gene is optionally ALDOB; whereinthe disease is glycogen storage disease type I and the defective gene isoptionally G6PC, SLC37A4, or SLC17A3; wherein the disease is glycogenstorage disease type II and the defective gene is optionally GM; whereinthe disease is glycogen storage disease type III and the defective geneis optionally AGL; wherein the disease is glycogen storage disease typeIV and the defective gene is optionally GBE1; wherein the disease isglycogen storage disease type V and the defective gene is optionallyPYGM; wherein the disease is glycogen storage disease type VI and thedefective gene is optionally PYGL; wherein the disease is glycogenstorage disease type VII and the defective gene is optionally PYGM;wherein the disease is glycogen storage disease type IX and thedefective gene is optionally PHKA1, PHKA2, PHKB, PHKG1, or PHKG2;wherein the disease is glycogen storage disease type XI and thedefective gene is optionally SLC2A2; wherein the disease is glycogenstorage disease type XII and the defective gene is optionally ALDOA;wherein the disease is glycogen storage disease type XIII and thedefective gene is optionally ENO1, ENO2, or ENO3; wherein the disease isglycogen storage disease type 0 and the defective gene is optionallyGYS1 or GYS2; wherein the disease is pyruvate carboxylase deficiency andthe defective gene is optionally PC; wherein the disease is pyruvatekinase deficiency and the defective gene is optionally PKLR; wherein thedisease is transaldolase deficiency and the defective gene is optionallyTALDO1; wherein the disease is triosephosphate isomerase deficiency andthe defective gene is optionally TPI1; wherein the disease is fructosebisphosphatase deficiency and the defective gene is optionally FBP1;wherein the disease is hyperoxaluria and the defective gene isoptionally AGXT or GRHPR; wherein the disease is hexokinase deficiencyand the defective gene is optionally HK1; wherein the disease isglucose-galactose malabsorption and the defective gene is optionallySLC5A1; or wherein the disease is glucose-6-phosphate dehydrogenasedeficiency and the defective gene is optionally G6PD.
 137. The method ofclaim 135, wherein the metabolic disorder is a disorder of amino acidmetabolism wherein the disease is alkaptonuria and the defective gene isoptionally HGD; wherein the disease is aspartylglucosaminuria and thedefective gene is optionally AGA; wherein the disease is methylmalonicacidemia and the defective gene is optionally MUT, MCEE, MMAA, MMAB,MMACHC, MMADHC, or LMBRD1; wherein the disease is maple syrup urinedisease and the defective gene is optionally BCKDHA, BCKDHB, DBT, orDLD; wherein the disease is homocystinuria and the defective gene isoptionally CBS; wherein the disease is tyrosinemia and the defectivegene is optionally FAH, TAT, or HPD; wherein the disease istrimethylaminuria and the defective gene is optionally FMO3; wherein thedisease is Hartnup disease and the defective gene is optionally SLC6A19;wherein the disease is biotinidase deficiency and the defective gene isoptionally BTD; wherein the disease is ornithine carbamoyltransferasedeficiency and the defective gene is optionally OTC; wherein the diseaseis carbamoyl-phosphate synthase I deficiency disease and the defectivegene is optionally CPS1; wherein the disease is citrullinemia and thedefective gene is optionally ASS or SLC25A13; wherein the disease ishyperargininemia and the defective gene is optionally ARG1; wherein thedisease is hyperhomocysteinemia and the defective gene is optionallyMTHFR; wherein the disease is hypermethioninemia and the defective geneis optionally MAT1A, GNMT, or AHCY; wherein the disease ishyperlysinemias and the defective gene is optionally AASS; wherein thedisease is nonketotic hyperglycinemia and the defective gene isoptionally GLDC, AMT, or GCSH; wherein the disease is Propionic acidemiaand the defective gene is optionally PCCA or PCCB; wherein the diseaseis hyperprolinemia and the defective gene is optionally ALDH4A1 orPRODH; wherein the disease is cystinuria and the defective gene isoptionally SLC3A1 or SLC7A9; wherein the disease is dicarboxylicaminoaciduria and the defective gene is optionally SLC1A1; wherein thedisease is glutaric acidemia type 2 and the defective gene is optionallyETFA, ETFB, or ETFDH; wherein the disease is isovaleric acidemia and thedefective gene is optionally IVD; or wherein the disease is2-hydroxyglutaric aciduria and the defective gene is optionally L2HGDHor D2HGDH.
 138. The method of claim 135, wherein the metabolic disorderis a disorder of the urea cycle wherein the disease is N-acetylglutamatesynthase deficiency and the defective gene is optionally NAGS; whereinthe disease is argininosuccinic aciduria and the defective gene isoptionally ASL; or wherein the disease is argininemia and the defectivegene is optionally ARG1.
 139. The method of claim 135, wherein themetabolic disorder is a disorder of fatty acid metabolism wherein thedisease is very long-chain acyl-coenzyme A dehydrogenase deficiency andthe defective gene is optionally ACADVL; wherein the disease islong-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency and thedefective gene is optionally HADHA; wherein the disease is medium-chainacyl-coenzyme A dehydrogenase deficiency and the defective gene isoptionally ACADM; wherein the disease is short-chain acyl-coenzyme Adehydrogenase deficiency and the defective gene is optionally ACADS;wherein the disease is 3-hydroxyacyl-coenzyme A dehydrogenase deficiencyand the defective gene is optionally HADH; wherein the disease is 2,4dienoyl-CoA reductase deficiency and the defective gene is optionallyNADK2; wherein the disease is 3-hydroxy-3-methylglutaryl-CoA lyasedeficiency and the defective gene is optionally HMGCL; wherein thedisease is malonyl-CoA decarboxylase deficiency and the defective geneis optionally MLYCD; wherein the disease is systemic primary carnitinedeficiency and the defective gene is optionally SLC22A5; wherein thedisease is carnitine-acylcarnitine translocase deficiency and thedefective gene is optionally SLC25A20; wherein the disease is carnitinepalmitoyltransferase I deficiency and the defective gene is optionallyCPT1A; wherein the disease is carnitine palmitoyltransferase IIdeficiency and the defective gene is optionally CPT2; wherein thedisease is lysosomal acid lipase deficiency and the defective gene isoptionally LIPA; or wherein the disease is Gaucher's disease and thedefective gene is optionally GBA.
 140. The method of claim 135, whereinthe metabolic disorder is a disorder of porphyrin metabolism wherein thedisease is acute intermittent porphyria and the defective gene isoptionally HMBS; wherein the disease is Gunther disease and thedefective gene is optionally UROS; wherein the disease is porphyriacutanea tarda and the defective gene is optionally UROD; wherein thedisease is hepatoerythropoietic porphyria and the defective gene isoptionally UROD; wherein the disease is hereditary coproporphyria andthe defective gene is optionally CPDX; wherein the disease is variegateporphyria and the defective gene is optionally PPDX; wherein the diseaseis erythropoietic protoporphyria and the defective gene is optionallyFECH; or wherein the disease is aminolevulinic acid dehydratasedeficiency porphyria and the defective gene is optionally ALAD.
 141. Themethod of claim 135, wherein the metabolic disorder is a disorder oflysosomal storage wherein the disease is Farber disease and thedefective gene is optionally ASAH1; wherein the disease is Krabbedisease and the defective gene is optionally GALC; wherein the diseaseis galactosialidosis and the defective gene is optionally CTSA; whereinthe disease is fabry disease and the defective gene is optionally GLA;wherein the disease is Schindler disease and the defective gene isoptionally NAGA; wherein the disease is GM1 gangliosidosis and thedefective gene is optionally GLB1; wherein the disease is Tay-Sachsdisease and the defective gene is optionally HEXA; wherein the diseaseis Sandhoff disease and the defective gene is optionally HEXB; whereinthe disease is GM2-gangliosidosis, AB variant and the defective gene isoptionally GM2A; wherein the disease is Niemann-Pick disease and thedefective gene is optionally SMPD1, NPC1, or NPC2; wherein the diseaseis metachromatic leukodystrophy and the defective gene is optionallyARSA or PSAP; wherein the disease is multiple sulfatase deficiency andthe defective gene is optionally SUMF1; wherein the disease is Hurlersyndrome and the defective gene is optionally IDUA; wherein the diseaseis Hunter syndrome and the defective gene is optionally IDS; wherein thedisease is Sanfilippo syndrome and the defective gene is optionallySGSH, NAGLU, HGSNAT, or GNS; wherein the disease is Morquio syndrome andthe defective gene is optionally GALNS or GLB1; wherein the disease isMaroteaux-Lamy syndrome and the defective gene is optionally ARSB;wherein the disease is Sly syndrome and the defective gene is optionallyGUSB; wherein the disease is sialidosis and the defective gene isoptionally NEU1, NEU2, NEU3, or NEU4; wherein the disease is I-celldisease and the defective gene is optionally GNPTAB or GNPTG; whereinthe disease is mucolipidosis type IV and the defective gene isoptionally MCOLN1; wherein the disease is infantile neuronal ceroidlipofuscinosis and the defective gene is optionally PPT1 or PPT2;wherein the disease is Jansky-Bielschowsky disease and the defectivegene is optionally TPP1; wherein the disease is Batten disease and thedefective gene is optionally CLN1, CLN2, CLN3, CLN5, CLN6, MFSD8, CLN8,or CTSD; wherein the disease is Kufs disease, Type A and the defectivegene is optionally CLN6 or PPT1; wherein the disease is Kufs disease,Type B and the defective gene is optionally DNAJC5 or CTSF; wherein thedisease is alpha-mannosidosis and the defective gene is optionallyMAN2B1, MAN2B2, or MAN2C1; wherein the disease is beta-mannosidosis andthe defective gene is optionally MANBA; wherein the disease isfucosidosis and the defective gene is optionally FUCA1; wherein thedisease is cystinosis and the defective gene is optionally CTNS; whereinthe disease is pycnodysostosis and the defective gene is optionallyCTSK; wherein the disease is Salla disease and the defective gene isoptionally SLC17A5; wherein the disease is Infantile free sialic acidstorage disease and the defective gene is optionally SLC17A5; or whereinthe disease is Danon disease and the defective gene is optionally LAMP2.142. The method of claim 135, wherein the metabolic disorder is adisorder of peroxisome biogenesis wherein the disease is Zellwegersyndrome and the defective gene is optionally PEX1, PEX2, PEX3, PEX5,PEX6, PEX12, PEX14, or PEX26; wherein the disease is Infantile Refsumdisease and the defective gene is optionally PEX1, PEX2, or PEX26;wherein the disease is neonatal adrenoleukodystrophy and the defectivegene is optionally PEX5, PEX1, PEX10, PEX13, or PEX26; wherein thedisease is RCDP Type 1 and the defective gene is optionally PEX7;wherein the disease is pipecolic acidemia and the defective gene isoptionally PAHX; wherein the disease is acatalasia and the defectivegene is optionally CAT; wherein the disease is hyperoxaluria type 1 andthe defective gene is optionally AGXT; wherein the disease is Acyl-CoAoxidase deficiency and the defective gene is optionally ACOX1; whereinthe disease is D-bifunctional protein deficiency and the defective geneis optionally HSD17B4; wherein the disease is dihydroxyacetonephosphateacyltransferase deficiency and the defective gene is optionally GNPAT;wherein the disease is X-linked adrenoleukodystrophy and the defectivegene is optionally ABCD1; wherein the disease is α-methylacyl-CoAracemase deficiency and the defective gene is optionally AMACR; whereinthe disease is RCDP Type 2 and the defective gene is optionally DHAPAT;wherein the disease is RCDP Type 3 and the defective gene is optionallyAGPS; wherein the disease is adult refsum disease-1 and the defectivegene is optionally PHYH; or wherein the disease is mulibrey nanism andthe defective gene is optionally TRIM37.
 143. The method of claim 135,wherein the metabolic disorder is a disorder of purine or pyrimidinemetabolism wherein the disease is Lesch-Nyhan syndrome and the defectivegene is optionally HPRT; wherein the disease is adeninephosphoribosyltransferase deficiency and the defective gene isoptionally APRT; wherein the disease is adenosine deaminase deficiencyand the defective gene is optionally ADA; wherein the disease isAdenosine monophosphate deaminase deficiency type 1 and the defectivegene is optionally AMPD1; wherein the disease is adenylosuccinate lyasedeficiency and the defective gene is optionally ADSL; wherein thedisease is dihydropyrimidine dehydrogenase deficiency and the defectivegene is optionally DPYD; wherein the disease is Miller syndrome and thedefective gene is optionally DHODH; wherein the disease is oroticaciduria and the defective gene is optionally UMPS; wherein the diseaseis purine nucleoside phosphorylase deficiency and the defective gene isoptionally PNP; or wherein the disease is xanthinuria and the defectivegene is optionally XDH, MOCS1, or MOCS2, GEPH.
 144. A method formodulating transthyretin (TTR), comprising administering an effectiveamount of a synthetic RNA encoding TTR to a subject, wherein thesynthetic RNA comprises one or more non-canonical nucleotides that avoidsubstantial cellular toxicity.
 145. The method of claim 144, wherein themodulating results in an increase in the quantity of TTR in the subject.146. The method of claim 144, wherein the modulating results in adecrease in the quantity of TTR in the subject.
 147. The method of anyone of claims 144 to 146, wherein the modulating results in treatment ofone or more of an amyloid disease, senile systemic amyloidosis (SSA),familial amyloid polyneuropathy (FAP), and familial amyloidcardiomyopathy (FAC).
 148. The method of any one of the above claims,wherein the non-canonical nucleotides have one or more substitutions atpositions selected from the 2C, 4C, and 5C positions for a pyrimidine,or selected from the 6C, 7N and 8C positions for a purine.
 149. Themethod of any one of the above claims, wherein the non-canonicalnucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine,5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine,5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine,5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine,5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridine, optionally at anamount of at least 50%, or at least 60%, or at least 70%, or at least80%, or at least 90%, or 100% of the non-canonical nucleotides.
 150. Themethod of any one of the above claims, wherein at least about 50% ofcytidine residues are non-canonical nucleotides, and which are selectedfrom 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine,5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.
 151. Themethod of any one of the above claims, wherein at least about 75% or atleast about 90% of cytidine residues are non-canonical nucleotides, andthe non-canonical nucleotides are selected from 5-hydroxycytidine,5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine,5-formylcytidine, and 5-methoxycytidine.
 152. The method of any one ofthe above claims, wherein at least about 20% of uridine, or at leastabout 40%, or at least about 50%, or at least about 75%, or at aboutleast 90% of uridine residues are non-canonical nucleotides, and thenon-canonical are selected from pseudouridine, 5-hydroxyuridine,5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine,5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine,5-methylpseudouridine, 5-hydroxymethylpseudouridine,5-carboxypseudouridine, 5-formylpseudouridine, and5-methoxypseudouridine.
 153. The method of any one of the above claims,wherein at least about 40%, or at least about 50%, or at least about75%, or at about least 90% of uridine residues are non-canonicalnucleotides, and the non-canonical nucleotides are selected frompseudouridine, 5-hydroxyuridine, 5-methyluridine,5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine,5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-hydroxymethylpseudouridine, 5-carboxypseudouridine,5-formylpseudouridine, and 5-methoxypseudouridine.
 154. The method ofany one of the above claims, wherein at least about 10% of guanineresidues are non-canonical nucleotides, and the non-canonical nucleotideis optionally 7-deazaguanosine.
 155. The method of any one of the aboveclaims, wherein the synthetic RNA contains no more than about 50%7-deazaguanosine in place of guanosine residues.
 156. The method of anyone of the above claims, wherein the synthetic RNA does not containnon-canonical nucleotides in place of adenosine residues.
 157. Themethod of any one of the above claims, wherein the synthetic RNAcomprises a 5′ cap structure.
 158. The method of any one of the aboveclaims, wherein the synthetic RNA 5′-UTR comprises a Kozak consensussequence.
 159. The method of any one of the above claims, wherein thesynthetic RNA 5′-UTR comprises a sequence that increases RNA stabilityin vivo, and the 5′-UTR may comprise an alpha-globin or beta-globin5′-UTR.
 160. The method of any one of the above claims, wherein thesynthetic RNA 3′-UTR comprises a sequence that increases RNA stabilityin vivo, and the 3′-UTR may comprise an alpha-globin or beta-globin3′-UTR.
 161. The method of any one of the above claims, wherein thesynthetic RNA comprises a 3′ poly(A) tail.
 162. The method of any one ofthe above claims, wherein the synthetic RNA 3′ poly(A) tail is fromabout 20 nucleotides to about 250 nucleotides in length.
 163. The methodof any one of the above claims, wherein the synthetic RNA is from about200 nucleotides to about 5000 nucleotides in length.
 164. The method ofany one of the above claims, wherein the synthetic RNA is from about 500to about 2000 nucleotides in length, or about 500 to about 1500nucleotides in length, or about 500 to about 1000 nucleotides in length.165. The method of any one of the above claims, wherein the syntheticRNA is prepared by in vitro transcription.
 166. The method of any one ofthe above claims, wherein the effective amount of the synthetic RNA isadministered as one or more injections containing about 10 ng to about5000 ng of RNA.
 167. The method of any one of the above claims, whereinthe effective amount of the synthetic RNA is administered as one or moreinjections each containing no more than about 10 ng, or no more thanabout 20 ng, or no more than about 50 ng, or no more than about 100 ng,or no more than about 200 ng, or no more than about 300 ng, or no morethan about 400 ng, or no more than about 500 ng, or no more than about600 ng, or no more than about 700 ng, or no more than about 800 ng, orno more than about 900 ng, or no more than about 1000 ng, or no morethan about 1100 ng, or no more than about 1200 ng, or no more than about1300 ng, or no more than about 1400 ng, or no more than about 1500 ng,or no more than about 1600 ng, or no more than about 1700 ng, or no morethan about 1800 ng, or no more than about 1900 ng, or no more than about2000 ng, or no more than about 3000 ng, or no more than about 4000 ng,or no more than about 5000 ng.
 168. The method of any one of the aboveclaims, wherein the effective amount of the synthetic RNA isadministered as one or more injections each containing about 10 ng, orabout 20 ng, or about 50 ng, or about 100 ng, or about 200 ng, or about300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000ng.
 169. The method of any one of the above claims, wherein theeffective amount of the synthetic RNA comprises one or more lipids toenhance uptake of RNA by cells.
 170. The method of any one of the aboveclaims, wherein the effective amount of the synthetic RNA comprises acationic liposome formulation and the lipids are optionally selectedfrom Table
 1. 171. The method of any one of the above claims, whereinthe subject is a human.
 172. The method of any one of the above claims,wherein the effective amount of the synthetic RNA is administered aboutweekly, for at least 2 weeks.
 173. The method of any one of the aboveclaims, wherein the effective amount of the synthetic RNA isadministered about every other week for at least one month.
 174. Themethod of any one of the above claims, wherein the effective amount ofthe synthetic RNA is administered monthly or about every other month.175. The method of any one of the above claims, wherein the effectiveamount of the synthetic RNA is administered for at least two months, orat least 4 months, or at least 6 months, or at least 9 months, or atleast one year.
 176. A composition comprising an effective amount of thesynthetic RNA used in any one of the above claims.
 177. A pharmaceuticalcomposition, comprising the composition of claim 176 and apharmaceutically acceptable excipient.
 178. Use of the composition ofclaim 176 or the pharmaceutical composition of claim 177 in thetreatment of a disease or disorder described herein.
 179. Use of thecomposition of claim 176 or the pharmaceutical composition of claim 177in the manufacture of a medicament for the treatment of a disease ordisorder described herein.